Immunoconjugates Comprising Anti-HER2 Antibodies and Pyrrolobenzodiazepines

ABSTRACT

The invention provides immunoconjugates comprising anti-HER2 antibodies and methods of using the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.15/438,418, filed Feb. 21, 2017, which is a continuation ofInternational Application No. PCT/US2015/050382, filed Sep. 16, 2015,which claims the benefit of priority of U.S. Provisional Application No.62/051,562, filed Sep. 17, 2014, each of which is incorporated byreference herein in its entirety for any purpose.

SEQUENCE LISTING

The present application is filed with a Sequence Listing in electronicformat. The Sequence Listing is provided as a file entitled“2019-04-22_01146-0039-01US_Sequence_Listing_ST25.txt” created on Feb.14, 2017, which is 76,260 bytes in size. The information in theelectronic format of the sequence listing is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to immunoconjugates comprising anti-HER2antibodies and methods of using the same.

BACKGROUND

Breast cancer is a highly significant cause of morbidity and mortalityworldwide. There are over 1.3 million cases of breast cancer diagnosedglobally each year with more than 450,000 deaths related to the disease(Jemal A, Bray F, Center M, et al. Global cancer statistics. CA Cancer JClin, 2011; 61(2):69-90).

The HER2 (ErbB2) receptor tyrosine kinase is a member of the epidermalgrowth factor receptor (EGFR) family of transmembrane receptors.Overexpression of HER2 is observed in approximately 20% of human breastcancers and is implicated in the aggressive growth and poor clinicaloutcomes associated with these tumors (Slamon et al (1987) Science235:177-182). HER2 protein overexpression can be determined using animmunohistochemistry based assessment of fixed tumor blocks (Press M F,et al (1993) Cancer Res 53:4960-70).

Trastuzumab (CAS 180288-69-1, HERCEPTIN®, huMAb4D5-8, rhuMAb HER2,Genentech) is a recombinant DNA-derived, IgG1 kappa, monoclonal antibodythat is a humanized version of a murine anti-HER2 antibody (4D5) thatselectively binds with high affinity in a cell-based assay (Kd=5 nM) tothe extracellular domain of HER2 (U.S. Pat. Nos. 5,677,171; 5,821,337;6,054,297; 6,165,464; 6,339,142; 6,407,213; 6,639,055; 6,719,971;6,800,738; 7,074,404; Coussens et al (1985) Science 230:1132-9; Slamonet al (1989) Science 244:707-12; Slamon et al (2001) New Engl. J. Med.344:783-792). Trastuzumab has been shown, in both in vitro assays and inanimals, to inhibit the proliferation of human tumor cells thatoverexpress HER2 (Hudziak et al (1989) Mol Cell Biol 9:1165-72; Lewis etal (1993) Cancer Immunol Immunother; 37:255-63; Baselga et al (1998)Cancer Res. 58:2825-2831). Trastuzumab is a mediator ofantibody-dependent cellular cytotoxicity, ADCC (Lewis et al (1993)Cancer Immunol Immunother 37(4):255-263; Hotaling et al (1996)[abstract]. Proc. Annual Meeting Am Assoc Cancer Res; 37:471; Pegram MD, et al (1997) [abstract]. Proc Am Assoc Cancer Res; 38:602; Sliwkowskiet al (1999) Seminars in Oncology 26(4), Suppl 12:60-70; Yarden Y. andSliwkowski, M. (2001) Nature Reviews: Molecular Cell Biology, MacmillanMagazines, Ltd., Vol. 2:127-137).

HERCEPTIN® was approved in 1998 for the treatment of patients withHER2-overexpressing metastatic breast cancers (Baselga et al, (1996) J.Clin. Oncol. 14:737-744) that have received extensive prior anti-cancertherapy, and has since been used in over 300,000 patients (Slamon D J,et al. N Engl J Med 2001; 344:783-92; Vogel C L, et al. J Clin Oncol2002; 20:719-26; Marty M, et al. J Clin Oncol 2005; 23:4265-74; Romond EH, et al. T N Engl J Med 2005; 353:1673-84; Piccart-Gebhart M J, et al.N Engl J Med 2005; 353:1659-72; Slamon D, et al. [abstract]. BreastCancer Res Treat 2006, 100 (Suppl 1): 52). In 2006, the FDA approvedHERCEPTIN® (trastuzumab, Genentech Inc.) as part of a treatment regimencontaining doxorubicin, cyclophosphamide and paclitaxel for the adjuvanttreatment of patients with HER2-positive, node-positive breast cancer.

Trastuzumab-MCC-DM1 (T-DM1, trastuzumab emtansine, ado-trastuzumabemtansine, KADCYLA®), a novel antibody-drug conjugate (ADC) for thetreatment of HER2-positive breast cancer, is composed of the cytotoxicagent DM1 (a thiol-containing maytansinoid anti-microtubule agent)conjugated to trastuzumab at lysine side chains via an MCC linker, withan average drug load (drug to antibody ratio) of about 3.5. Afterbinding to HER2 expressed on tumor cells, T-DM1 undergoesreceptor-mediated internalization, resulting in intracellular release ofcytotoxic catabolites containing DM1 and subsequent cell death.

The U.S. Food and Drug Administration approved ado-trastuzumabemtansine, marketed under the tradename KADCYLA®, on Feb. 22, 2013 forthe treatment of patients with HER2-positive, metastatic breast cancerwho previously received treatment with trastuzumab and a taxane.

Pertuzumab (also known as recombinant humanized monoclonal antibody 2C4,rhuMAb 2C4, PERJETA®, Genentech, Inc, South San Francisco) representsthe first in a new class of agents known as HER dimerization inhibitors(HDI) and functions to inhibit the ability of HER2 to form activeheterodimers or homodimers with other HER receptors (such as EGFR/HER1,HER2, HER3 and HER4). See, for example, Harari and Yarden Oncogene19:6102-14 (2000); Yarden and Sliwkowski. Nat Rev Mol Cell Biol 2:127-37(2001); Sliwkowski Nat Struct Biol 10:158-9 (2003); Cho et al. Nature421:756-60 (2003); and Malik et al. Pro Am Soc Cancer Res 44:176-7(2003)

Pertuzumab blockade of the formation of HER2-HER 3 heterodimers in tumorcells has been demonstrated to inhibit critical cell signaling, whichresults in reduced tumor proliferation and survival (Agus et al. CancerCell 2:127-37 (2002)).

Pertuzumab has been evaluated in Phase II studies in combination withtrastuzumab in patients with HER2-positive metastatic breast cancer whohave previously received trastuzumab for metastatic disease. One study,conducted by the National cancer Institute (NC1), enrolled 11 patientswith previously treated HER2-positive metastatic breast cancer. Two outof the 11 patients exhibited a partial response (PR) (Baselga et al., JClin Oncol 2007 ASCO Annual Meeting Proceedings; 25:18 S (June 20Supplement): 1004. The results of a Phase II neoadjuvant studyevaluating the effect of a novel combination regimen of pertuzumab andtrastuzumab plus chemotherapy (Docetaxel) in women with early-stageHER2-positive breast cancer, presented at the CTRC-AACR San AntonioBreast Cancer Symposium (SABCS), Dec. 8-12, 2010, showed that the twoHER2 antibodies plus Docetaxel given in the neoadjuvant setting prior tosurgery significantly improved the rate of complete tumor disappearance(pathological complete response rate, pCR, of 45.8 percent) in thebreast by more than half compared to trastuzumab plus Docetaxel (pCR of29.0 percent), p=0.014.

Pertuzumab, marketed under the tradename PERJETA®, was approved in 2012for the treatment of patients with advanced or late-stage (metastatic)HER2-positive breast cancer. HER2-positive breast cancers have increasedamounts of the HER2 protein that contributes to cancer cell growth andsurvival.

On Sep. 30, 2013, the U.S. Food and Drug Administration grantedaccelerated approval to PERJETA® (pertuzumab) as part of a completetreatment regimen for patients with early stage breast cancer (EBC)before surgery (neoadjuvant setting). PERJETA® is the first FDA-approveddrug for the neoadjuvant treatment of breast cancer.

There is a need in the art for additional safe and effective agents thattarget HER2 for treatment of HER2-associated conditions, such as breastcancer, for use in monotherapy and combination therapy. The inventionfulfills that need and provides other benefits.

SUMMARY

The invention provides immunoconjugates comprising anti-HER2 antibodiesand methods of using the same.

In some embodiments, an immunoconjugate is provided, which comprises anantibody and a cytotoxic agent, wherein the cytotoxic agent is acenter-linked pyrrolobenzodiazepine. In some embodiments, theimmunoconjugate has the formula Ab-(L-D)p, wherein:

-   -   a) Ab is the antibody of any one of claim 1 to 16;    -   b) L is a linker;    -   c) D is a center-linked pyrrolobenzodiazepine; and    -   d) p ranges from 1-8.        In some embodiments, L-D is of Formula A:

wherein:

R² is

where R^(36a) and R^(36b) are independently selected from H, F, C₁₋₄saturated alkyl, C₂₋₃ alkenyl, which alkyl and alkenyl groups areoptionally substituted by a group selected from C₁₋₄ alkyl amido andC₁₋₄ alkyl ester; or, when one of R^(36a) and R^(36b) is H, the other isselected from nitrile and a C₁₋₄ alkyl ester;

R⁶ and R⁹ are independently selected from H, R, OH, OR, SH, SR, NH₂,NHR, NRR′, NO₂, Me₃Sn and halo;

R⁷ is independently selected from H, R, OH, OR, SH, SR, NH₂, NHR, NRR′,NO₂, Me₃Sn and halo;

Y has the formula:

G is a linker connected to the antibody;

n is an integer selected in the range of 0 to 48;

R^(A4) is a C₁₋₆ alkylene group;

either:

-   -   (a) R¹⁰ is H, and R¹¹ is OH, OR^(A), where R^(A) is C₁₋₄ alkyl;        or    -   (b) R⁰ and R¹¹ form a nitrogen-carbon double bond between the        nitrogen and carbon atoms to which they are bound; or    -   (c) R⁰ is H and R¹¹ is OSO_(z)M, where z is 2 or 3 and M is a        monovalent pharmaceutically acceptable cation;

R and R′ are each independently selected from optionally substitutedC₁₋₁₂ alkyl, C₃₋₂₀ heterocyclyl and C₅₋₂₀ aryl groups, and optionally inrelation to the group NRR′, R and R′ together with the nitrogen atom towhich they are attached form an optionally substituted 4-, 5-, 6- or7-membered heterocyclic ring;

R⁶, R¹⁷, R¹⁹, R²⁰, R²¹ and R²² are as defined for R⁶, R⁷, R⁹, R¹⁰, R¹¹and R² respectively;

Z is CH or N;

T and T″ are independently selected from a single bond or a C₁₋₉alkylene, which chain may be interrupted by one or more heteroatoms,e.g., O, S, N(H), NMe, provided that the number of atoms in the shortestchain of atoms between X and X′ is 3 to 12 atoms; and

X and X′ are independently selected from O, S and N(H); and

wherein the antibody binds to HER2 and comprises (a) HVR-H1 comprisingthe amino acid sequence of SEQ ID NO: 15; (b) HVR-H2 comprising theamino acid sequence of SEQ ID NO: 16; (c) HVR-H3 comprising the aminoacid sequence of SEQ ID NO: 17; (d) HVR-L1 comprising the amino acidsequence of SEQ ID NO: 12; (e) HVR-L2 comprising the amino acid sequenceof SEQ ID NO: 13; and (f) HVR-L3 comprising the amino acid sequence ofSEQ ID NO: 14. In some embodiments, the antibody comprises a heavy chainvariable region comprising the sequence of SEQ ID NO: 11 and a lightchain variable region comprising the sequence of SEQ ID NO: 10. In someembodiments, the antibody is a monoclonal antibody. In some embodiments,the antibody is a humanized or chimeric antibody. In some embodiments,the antibody is an antibody fragment that binds HER2.

In some embodiments, HER2 is human HER2 comprising amino acids 23 to1255 of SEQ ID NO: 1. In some embodiments, the antibody binds toextracellular domain I of HER2. In some embodiments, extracellulardomain I of HER2 has the sequence of SEQ ID NO: 35. In some embodiments,the antibody binds to loop 163-189 and loop 185-189 of extracellulardomain I (e.g., a first loop defined by amino acids 163-189 and a secondloop defined by amino acids 185-189 of extracellular domain I). In someembodiments, the antibody contacts His171, Ser186, Ser187 and Glu188 ofextracellular domain I.

In some embodiments, the antibody is an IgG1, IgG2a or IgG2b antibody.In any of the embodiments described herein, the antibody may compriseone or more engineered free cysteine amino acids residues. In any of theembodiments described herein, the one or more engineered free cysteineamino acids residues may be located in the heavy chain. In any of theembodiments described herein, the one or more engineered free cysteineamino acids residues may be located in the light chain. In someembodiments, the antibody comprises at least one mutation in the heavychain constant region selected from A118C and S400C. In someembodiments, the antibody comprises at least one mutation in the lightchain constant region selected from K149C and V205C.

In some embodiments, the antibody comprises:

-   -   a) a heavy chain comprising the sequence of SEQ ID NO: 19 and a        light chain comprising the sequence of SEQ ID NO: 18; or    -   b) a heavy chain comprising the sequence of SEQ ID NO: 19 and a        light chain comprising the sequence of SEQ ID NO: 23; or    -   c) a heavy chain comprising the sequence of SEQ ID NO: 24 and a        light chain comprising the sequence of SEQ ID NO: 18.

In some embodiments, the antibody comprises the heavy chain constantregion of SEQ ID NO: 28.

In some embodiments, the antibody comprises the light chain constantregion of SEQ ID NO: 25.

In some embodiments, the immunoconjugate comprises an antibody thatbinds to HER2, wherein the antibody comprises a heavy chain comprisingthe sequence of SEQ ID NO: 19 and a light chain comprising the sequenceof SEQ ID NO: 23. In some embodiments, an isolated antibody that bindsto HER2 is provided, wherein the antibody comprises a heavy chaincomprising the sequence of SEQ ID NO: 24 and a light chain comprisingthe sequence of SEQ ID NO: 18.

In some embodiments of the immunoconjugate, R⁹ is H. In someembodiments, R⁶ is H. In some embodiments, R⁷ is OR^(7A), where R^(7A)is optionally substituted C₁₋₄ alkyl. In some embodiments, R^(7A) is Me.In some embodiments, X is O. In some embodiments, T is selected from asingle bond, C₁, and a C₂ alkylene group. In some embodiments, T is a C₁alkylene group. In some embodiments, R^(36a) and R^(36b) are both H. Insome embodiments, R^(36a) and R^(36b) are both methyl. In someembodiments, one of R^(36a) and R^(36b) is H, and the other is selectedfrom C₁₋₄ saturated alkyl, C₂₋₃ alkenyl, which alkyl and alkenyl groupsare optionally substituted. In some embodiments, the group of R^(36a)and R^(36b) which is not H is selected from methyl and ethyl. In someembodiments, R¹⁰ is H, and R¹¹ is OH. In some embodiments, R¹⁰ and R¹¹form a nitrogen-carbon double bond between the nitrogen and carbon atomsto which they are bound. In some embodiments, R¹⁶, R¹⁷, R¹⁹, R²⁰, R²¹,R²², X′ and T′ are the same as R⁶, R⁷, R⁹, R¹⁰, R¹¹, R², X and T,respectively.

In some embodiments, an immunoconjugate comprises the structure:

wherein Y is defined as above. In some embodiments, the immunoconjugatecomprises the structure:

wherein Ab is an antibody that binds HER2 described herein.

In any of the immunoconjugates described herein, p may range from 1.3-2,1.4-2, 1.5-2, or 2-5.

In some embodiments, a pharmaceutical formulation is provided,comprising an immunoconjugate described herein and a pharmaceuticallyacceptable carrier. In some embodiments, the pharmaceutical formulationfurther comprises an additional therapeutic agent. In some embodiments,the additional therapeutic agent is an antibody or immunoconjugate thatbinds to HER2. In some embodiments, the additional therapeutic agent is(i) an antibody or immunoconjugate that binds to domain II of HER2,and/or (ii) an antibody or immunoconjugate that binds to domain IV orHER2. In some embodiments, the additional therapeutic agent is (i) anantibody or immunoconjugate that binds to epitope 2C4, and/or (ii) anantibody or immunoconjugate that binds to epitope 4D5. In someembodiments, the additional therapeutic agent is selected fromtrastuzumab, trastuzumab-MCC-DM1 (T-DM1), and pertuzumab. In someembodiments, the pharmaceutical formulation further comprises (1)trastuzumab or T-DM1, and (2) pertuzumab.

In some embodiments, methods of treating an individual having aHER2-positive cancer are provided. In some embodiments, a methodcomprises administering to the individual an effective amount of animmunoconjugate described herein, or a pharmaceutical compositiondescribed herein. In some embodiments, the HER2-positive cancer isbreast cancer or gastric cancer. In some embodiments, the HER2-positivebreast cancer is early-stage breast cancer. In some embodiments, theHER2-positive breast cancer is metastatic breast cancer. In someembodiments, the HER2-positive cancer is recurrent cancer. In someembodiments, the recurrent cancer is locally recurrent cancer. In someembodiments, the HER2-positive cancer is advanced cancer. In someembodiments, the HER2-positive cancer is non-resectable. In someembodiments, the method further comprises administering an additionaltherapeutic agent to the individual.

In some embodiments, a method of treating an individual having aHER2-positive cancer comprises administering to the individual aneffective amount of an immunoconjugate described herein and at least oneadditional therapeutic agent to the individual. In some embodiments, theadditional therapeutic agent is an antibody or immunoconjugate thatbinds to HER2. In some embodiments, the additional therapeutic agent is(i) an antibody or immunoconjugate that binds to domain II of HER2,and/or (ii) an antibody or immunoconjugate that binds to domain IV orHER2. In some embodiments, the additional therapeutic agent is (i) anantibody or immunoconjugate that binds to epitope 2C4, and/or (ii) anantibody or immunoconjugate that binds to epitope 4D5. In someembodiments, the additional therapeutic agent is selected fromtrastuzumab, trastuzumab-MCC-DM1 (T-DM1), and pertuzumab. In someembodiments, the additional therapeutic agents are (1) trastuzumab orT-DM1, and (2) pertuzumab. In some embodiments, the HER2-positive canceris breast cancer or gastric cancer. In some embodiments, theHER2-positive breast cancer is early-stage breast cancer. In someembodiments, the HER2-positive breast cancer is metastatic breastcancer. In some embodiments, the HER2-positive cancer is recurrentcancer. In some embodiments, the recurrent cancer is locally recurrentcancer. In some embodiments, the HER2-positive cancer is advancedcancer. In some embodiments, the HER2-positive cancer is non-resectable.

In some embodiments, a method of treating an individual having aHER2-positive cancer comprises:

-   -   a) subjecting the individual to neoadjuvant treatment with an        immunoconjugate described herein or a pharmaceutical formulation        described herein,    -   b) removing the cancer by definitive surgery, and    -   c) subjecting the individual to adjuvant treatment with an        immunoconjugate described herein or a pharmaceutical formulation        described herein.

In some embodiments, the HER2-positive cancer is breast cancer orgastric cancer.

In some embodiments, methods of inhibiting proliferation of aHER2-positive cell are provided. In some embodiments a method comprisesexposing the cell to an immunoconjugate described herein underconditions permissive for binding of the immunoconjugate to HER2 on thesurface of the cell, thereby inhibiting proliferation of the cell. Insome embodiments, the cell is a breast cancer cell of a gastric cancercell.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows an alignment of the human VH subgroup I (VH_(I)) consensussequence and heavy chain variable region sequences of murine 7C2.B9(“7C2”) and humanized 7C2.v2.2.LA, as described in Example 1.

FIG. 2 shows an alignment of the human VL kappa IV (VL_(KIV)) consensussequence and light chain variable region sequences of murine 7C2.B9(“7C2”) and humanized 7C2.v2.2.LA, as described in Example 1.

FIG. 3 shows the Her2 extracellular domain structure, with domains I toIV indicated, and the domains to which anti-Her2 antibodies trastuzumab,pertuzumab, and 7C2 bind.

FIG. 4 shows change in tumor volume (mm3) over time upon treatment withhu7C2.v2.2.LA antibody-drug conjugates (ADCs), as described in Example3.

FIG. 5 shows change in tumor volume (mm3) over time upon treatment withhu7C2.v2.2.LA antibody-drug conjugates (ADCs), as described in Example4.

FIG. 6 show the structures for certain antibody-drug conjugates used inthe examples herein.

FIGS. 7A-B show the pertuzumab main species antibody light chain (A) andheavy chain (B) amino acid sequences.

FIGS. 8A-B show exemplary pertuzumab variant species antibody lightchain (A) and heavy chain (B) amino acid sequences.

FIGS. 9A-B show the trastuzumab antibody light chain (A) and heavy chain(B) amino acid sequences.

FIG. 10 shows a schematic of the Her2 receptor and the sequences fordomains I to IV.

FIGS. 11A-D show (A) crystal structure of the complex between HER2 ECD(surface shaded by domain and shown as a space-filling model) and 7C2Fab. The 7C2 Fab binds to domain I of HER2, which is different from thebinding epitopes of the trastuzumab Fab (Tmab, PDB code: 1N8Z) and thepertuzumab Fab (Pmab, PDB code: 1S78). (B) Superposition of thestructures of HER2 ECD within the trastuzumab/HER2 complex,pertuzumab/HER2 complex, and 7C2/HER2 complex. (C) The 7C2/HER2 complexinterface. The side chains of the residues involved in the 7C2/HER2interaction are shown as sticks. Some of the potential intermolecularhydrogen bonds are shown as dashed lines. (D) The 7C2 binding epitope ispartially overlapped with the chA21 single-chain Fv (scFv).Superposition of the structure of the chA21 scFv/HER2 complex (PDB code:3H3B) with the 7C2/HER2 complex.

DETAILED DESCRIPTION

Reference will now be made in detail to certain embodiments of theinvention, examples of which are illustrated in the accompanyingstructures and formulas. While the invention will be described inconjunction with the enumerated embodiments, it will be understood thatthey are not intended to limit the invention to those embodiments. Onthe contrary, the invention is intended to cover all alternatives,modifications, and equivalents which may be included within the scope ofthe present invention as defined by the claims. One skilled in the artwill recognize many methods and materials similar or equivalent to thosedescribed herein, which could be used in the practice of the presentinvention. The present invention is in no way limited to the methods andmaterials described.

All references cited throughout the disclosure are expresslyincorporated by reference herein in their entirety. In the event thatone or more of the incorporated literature, patents, and similarmaterials differs from or contradicts this application, including butnot limited to defined terms, term usage, described techniques, or thelike, this application controls.

I. DEFINITIONS

The words “comprise,” “comprising,” “include,” “including,” and“includes” when used in this specification and claims are intended tospecify the presence of stated features, integers, components, or steps,but they do not preclude the presence or addition of one or more otherfeatures, integers, components, steps, or groups thereof.

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencechanges. In some embodiments, the number of amino acid changes are 10 orless, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less,3 or less, or 2 or less. In some embodiments, the VL acceptor humanframework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

“Affinity” refers to the strength of the sum total of noncovalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., antibody and antigen). The affinity of a molecule Xfor its partner Y can generally be represented by the dissociationconstant (Kd). Affinity can be measured by common methods known in theart, including those described herein. Specific illustrative andexemplary embodiments for measuring binding affinity are described inthe following.

An “affinity matured” antibody refers to an antibody with one or morealterations in one or more hypervariable regions (HVRs), compared to aparent antibody which does not possess such alterations, suchalterations resulting in an improvement in the affinity of the antibodyfor antigen.

The terms “anti-HER2 antibody” and “an antibody that binds to HER2”refer to an antibody that is capable of binding HER2 with sufficientaffinity such that the antibody is useful as a diagnostic and/ortherapeutic agent in targeting HER2. In one embodiment, the extent ofbinding of an anti-HER2 antibody to an unrelated, non-HER2 protein isless than about 10% of the binding of the antibody to HER2 as measured,e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibodythat binds to HER2 has a dissociation constant (Kd) of ≤1 μM, ≤100 nM,≤10 nM, ≤5 nm, ≤4 nM, ≤3 nM, ≤2 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001nM (e.g., 10⁻⁸ M or less, e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ Mto 10⁻¹³ M). In certain embodiments, an anti-HER2 antibody binds to anepitope of HER2 that is conserved among HER2 from different species.

The term “antibody” is used herein in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody and that bindsthe antigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂; diabodies; linear antibodies; single-chain antibody molecules(e.g. scFv); and multispecific antibodies formed from antibodyfragments.

An “antibody that binds to the same epitope” as a reference antibodyrefers to an antibody that blocks binding of the reference antibody toits antigen in a competition assay by 50% or more, and conversely, thereference antibody blocks binding of the antibody to its antigen in acompetition assay by 50% or more. An exemplary competition assay isprovided herein.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth/proliferation. Examples of cancer include, butare not limited to, carcinoma, lymphoma, blastoma, sarcoma, andleukemia. In some embodiments, the cancer is breast cancer or gastriccancer. In some embodiments, a cancer is any HER2-positive cancer.

A “HER2-positive” cancer comprises cancer cells which have higher thannormal levels of HER2. Examples of HER2-positive cancer includeHER2-positive breast cancer and HER2-positive gastric cancer.Optionally, HER2-positive cancer has an immunohistochemistry (IHC) scoreof 2+ or 3+ and/or an in situ hybridization (ISH) amplification ratio≥2.0.

The term “early stage breast cancer (EBC)” or “early breast cancer” isused herein to refer to breast cancer that has not spread beyond thebreast or the axillary lymph nodes. This includes ductal carcinoma insitu and stage I, stage IIA, stage IIB, and stage IIIA breast cancers.

Reference to a tumor or cancer as a “Stage 0,” “Stage I,” “Stage II,”“Stage III,” or “Stage IV”, and various sub-stages within thisclassification, indicates classification of the tumor or cancer usingthe Overall Stage Grouping or Roman Numeral Staging methods known in theart. Although the actual stage of the cancer is dependent on the type ofcancer, in general, a Stage 0 cancer is an in situ lesion, a Stage Icancer is small localized tumor, a Stage II and III cancer is a localadvanced tumor which exhibits involvement of the local lymph nodes, anda Stage IV cancer represents metastatic cancer. The specific stages foreach type of tumor is known to the skilled clinician.

The term “metastatic breast cancer” means the state of breast cancerwhere the cancer cells are transmitted from the original site to one ormore sites elsewhere in the body, by the blood vessels or lymphatics, toform one or more secondary tumors in one or more organs besides thebreast.

An “advanced” cancer is one which has spread outside the site or organof origin, either by local invasion or metastasis. Accordingly, the term“advanced” cancer includes both locally advanced and metastatic disease.

A “recurrent” cancer is one which has regrown, either at the initialsite or at a distant site, after a response to initial therapy, such assurgery.

A “locally recurrent” cancer is cancer that returns after treatment inthe same place as a previously treated cancer.

An “operable” or “resectable” cancer is cancer which is confined to theprimary organ and suitable for surgery (resection).

A “non-resectable” or “unresectable” cancer is not able to be removed(resected) by surgery.

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ, respectively.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents a cellular function and/or causes cell death ordestruction. Cytotoxic agents include, but are not limited to,radioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³,Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu); chemotherapeuticagents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids(vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycinC, chlorambucil, daunorubicin or other intercalating agents); growthinhibitory agents; enzymes and fragments thereof such as nucleolyticenzymes; antibiotics; toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof; and the variousantitumor or anticancer agents disclosed below.

“Effector functions” refer to those biological activities attributableto the Fc region of an antibody, which vary with the antibody isotype.Examples of antibody effector functions include: Clq binding andcomplement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation.

An “effective amount” of an agent, e.g., a pharmaceutical formulation,refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result.The effective amount of the drug for treating cancer may reduce thenumber of cancer cells; reduce the tumor size; inhibit (i.e., slow tosome extent and preferably stop) cancer cell infiltration intoperipheral organs; inhibit (i.e., slow to some extent and preferablystop) tumor metastasis; inhibit, to some extent, tumor growth; and/orrelieve to some extent one or more of the symptoms associated with thecancer. To the extent the drug may prevent growth and/or kill existingcancer cells, it may be cytostatic and/or cytotoxic. The effectiveamount may extend progression free survival (e.g. as measured byResponse Evaluation Criteria for Solid Tumors, RECIST, or CA-125changes), result in an objective response (including a partial response,PR, or complete response, CR), increase overall survival time, and/orimprove one or more symptoms of cancer (e.g. as assessed by FOSI).

The term “epitope” refers to the particular site on an antigen moleculeto which an antibody binds.

The “epitope 4D5” or “4D5 epitope” or “4D5” is the region in theextracellular domain of HER2 to which the antibody 4D5 (ATCC CRL 10463)and trastuzumab bind. This epitope is close to the transmembrane domainof HER2, and within domain IV of HER2. To screen for antibodies whichbind to the 4D5 epitope, a routine cross-blocking assay such as thatdescribed in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed.Alternatively, epitope mapping can be performed to assess whether theantibody binds to the 4D5 epitope of HER2 (e.g. any one or more residuesin the region from about residue 550 to about residue 610, inclusive, ofHER2 (SEQ ID NO: 39).

The “epitope 2C4” or “2C4 epitope” is the region in the extracellulardomain of HER2 to which the antibody 2C4 binds. In order to screen forantibodies which bind to the 2C4 epitope, a routine cross-blocking assaysuch as that described in Antibodies, A Laboratory Manual, Cold SpringHarbor Laboratory, Ed Harlow and David Lane (1988), can be performed.Alternatively, epitope mapping can be performed to assess whether theantibody binds to the 2C4 epitope of HER2.

Epitope 2C4 comprises residues from domain II in the extracellulardomain of HER2. The 2C4 antibody and pertuzumab bind to theextracellular domain of HER2 at the junction of domains I, II and III(Franklin et al. Cancer Cell 5:317-328 (2004)).

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc regions andvariant Fc regions. In one embodiment, a human IgG heavy chain Fc regionextends from Cys226, or from Pro230, to the carboxyl-terminus of theheavy chain. However, the C-terminal lysine (Lys447) of the Fc regionmay or may not be present. Unless otherwise specified herein, numberingof amino acid residues in the Fc region or constant region is accordingto the EU numbering system, also called the EU index, as described inKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.,1991.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The terms “full length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

The term “glycosylated forms of HER2” refers to naturally occurringforms of HER2 that are post-translationally modified by the addition ofcarbohydrate residues.

The terms “host cell,” “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations.

Mutant progeny that have the same function or biological activity asscreened or selected for in the originally transformed cell are includedherein.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of Immunological Interest, FifthEdition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal., supra. In one embodiment, for the VH, the subgroup is subgroup IIIas in Kabat et al., supra.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization.

The term “hypervariable region” or “HVR,” as used herein, refers to eachof the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops (“hypervariable loops”).Generally, native four-chain antibodies comprise six HVRs; three in theVH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generallycomprise amino acid residues from the hypervariable loops and/or fromthe “complementarity determining regions” (CDRs), the latter being ofhighest sequence variability and/or involved in antigen recognition.Exemplary hypervariable loops occur at amino acid residues 26-32 (L1),50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3).(Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987).) Exemplary CDRs(CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acidresidues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 ofH2, and 95-102 of H3. (Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991).) With the exception of CDR1in VH, CDRs generally comprise the amino acid residues that form thehypervariable loops. CDRs also comprise “specificity determiningresidues,” or “SDRs,” which are residues that contact antigen. SDRs arecontained within regions of the CDRs called abbreviated-CDRs, or a-CDRs.Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, anda-CDR-H3) occur at amino acid residues 31-34 of L1, 50-55 of L2, 89-96of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3. (See Almagro andFransson, Front. Biosci. 13:1619-1633 (2008).) Unless otherwiseindicated, HVR residues and other residues in the variable domain (e.g.,FR residues) are numbered herein according to Kabat et al., supra.

An “immunoconjugate” is an antibody conjugated to one or moreheterologous molecule(s), including but not limited to a cytotoxicagent.

A “patient” or “individual” or “subject” is a mammal. Mammals include,but are not limited to, domesticated animals (e.g., cows, sheep, cats,dogs, and horses), primates (e.g., humans and non-human primates such asmonkeys), rabbits, and rodents (e.g., mice and rats). In certainembodiments, the patient, individual, or subject is a human. In someembodiments, the patient may be a “cancer patient,” i.e. one who issuffering or at risk for suffering from one or more symptoms of cancer,in particular gastric or breast cancer.

A “patient population” refers to a group of cancer patients. Suchpopulations can be used to demonstrate statistically significantefficacy and/or safety of a drug.

A “relapsed” patient is one who has signs or symptoms of cancer afterremission.

Optionally, the patient has relapsed after adjuvant or neoadjuvanttherapy.

A cancer or biological sample which “displays HER expression,amplification, or activation” is one which, in a diagnostic test,expresses (including overexpresses) a HER receptor, has amplified HERgene, and/or otherwise demonstrates activation or phosphorylation of aHER receptor.

“Neoadjuvant therapy” or “preoperative therapy” herein refers to therapygiven prior to surgery. The goal of neoadjuvant therapy is to provideimmediate systemic treatment, potentially eradicating micrometastasesthat would otherwise proliferate if the standard sequence of surgeryfollowed by systemic therapy were followed. Neoadjuvant therapy may alsohelp to reduce tumor size thereby allowing complete resection ofinitially unresectable tumors or preserving portions of the organ andits functions. Furthermore, neoadjuvant therapy permits an in vivoassessment of drug efficacy, which may guide the choice of subsequenttreatments.

“Adjuvant therapy” herein refers to therapy given after definitivesurgery, where no evidence of residual disease can be detected, so as toreduce the risk of disease recurrence. The goal of adjuvant therapy isto prevent recurrence of the cancer, and therefore to reduce the chanceof cancer-related death. Adjuvant therapy herein specifically excludesneoadjuvant therapy.

“Definitive surgery” is used as that term is used within the medicalcommunity. Definitive surgery includes, for example, procedures,surgical or otherwise, that result in removal or resection of the tumor,including those that result in the removal or resection of all grosslyvisible tumor. Definitive surgery includes, for example, complete orcurative resection or complete gross resection of the tumor. Definitivesurgery includes procedures that occur in one or more stages, andincludes, for example, multi-stage surgical procedures where one or moresurgical or other procedures are performed prior to resection of thetumor. Definitive surgery includes procedures to remove or resect thetumor including involved organs, parts of organs and tissues, as well assurrounding organs, such as lymph nodes, parts of organs, or tissues.Removal may be incomplete such that tumor cells might remain even thoughundetected.

“Survival” refers to the patient remaining alive, and includes diseasefree survival (DFS), progression free survival (PFS) and overallsurvival (OS). Survival can be estimated by the Kaplan-Meier method, andany differences in survival are computed using the stratified log-ranktest.

“Progression-Free Survival” (PFS) is the time from the first day oftreatment to documented disease progression (including isolated CNSprogression) or death from any cause on study, whichever occurs first.

“Disease free survival (DFS)” refers to the patient remaining alive,without return of the cancer, for a defined period of time such as about1 year, about 2 years, about 3 years, about 4 years, about 5 years,about 10 years, etc., from initiation of treatment or from initialdiagnosis. In one aspect, DFS is analyzed according to theintent-to-treat principle, i.e., patients are evaluated on the basis oftheir assigned therapy. The events used in the analysis of DFS caninclude local, regional and distant recurrence of cancer, occurrence ofsecondary cancer, and death from any cause in patients without a priorevent (e.g., breast cancer recurrence or second primary cancer).

“Overall survival” refers to the patient remaining alive for a definedperiod of time, such as about 1 year, about 2 years, about 3 years,about 4 years, about 5 years, about 10 years, etc., from initiation oftreatment or from initial diagnosis. In the studies underlying theinvention the event used for survival analysis was death from any cause.

By “extending survival” is meant increasing DFS and/or OS in a treatedpatient relative to an untreated patient, or relative to a controltreatment protocol. Survival is monitored for at least about six months,or at least about 1 year, or at least about 2 years, or at least about 3years, or at least about 4 years, or at least about 5 years, or at leastabout 10 years, etc., following the initiation of treatment or followingthe initial diagnosis.

By “monotherapy” is meant a therapeutic regimen that includes only asingle therapeutic agent for the treatment of the cancer or tumor duringthe course of the treatment period.

By “maintenance therapy” is meant a therapeutic regimen that is given toreduce the likelihood of disease recurrence or progression. Maintenancetherapy can be provided for any length of time, including extended timeperiods up to the life-span of the subject. Maintenance therapy can beprovided after initial therapy or in conjunction with initial oradditional therapies. Dosages used for maintenance therapy can vary andcan include diminished dosages as compared to dosages used for othertypes of therapy.

As defined herein, the terms “trastuzumab”, “HERCEPTIN®” and“huMAb4D5-8” are used interchangeably. Such antibody preferablycomprises the light and heavy chain amino acid sequences shown in SEQ IDNO: 30 and SEQ ID NO. 29, respectively.

For the purposes herein, “pertuzumab”, “PERJETA®” and “rhuMAb 2C4”, areused interchangeably. Such antibody comprises a main species antibodyhaving the light and heavy chain amino acid sequences in SEQ ID NOs: 32and 31, respectively (FIGS. 7A and B). In some embodiments, pertuzumabcomprises a variant species antibody with an amino-terminal leaderextension, e.g., comprising a light chain amino acid sequence of SEQ IDNO: 34, and a heavy chain amino acid sequence of SEQ ID NO: 33. Theantibody is optionally produced by recombinant Chinese Hamster Ovary(CHO) cells.

As defined herein, the terms “T-DM1,” “trastuzumab-MCC-DM1,”“ado-trastuzumab emtansine,” “trastuzumab emtansine,” and “KADCYLA®” areused interchangeably, and refer to trastuzumab linked through the linkermoiety MCC to the maytansinoid drug moiety DM1, including all mixturesof variously loaded and attached antibody-drug conjugates where 1, 2, 3,4, 5, 6, 7, and 8 drug moieties are covalently attached to the antibodytrastuzumab (U.S. Pat. Nos. 7,097,840; 8,337,856; US 2005/0276812; US2005/0166993).

An “isolated antibody” is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC). For review of methods for assessment of antibody purity, see,e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

An “isolated nucleic acid” refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

“Isolated nucleic acid encoding an anti-HER2 antibody” refers to one ormore nucleic acid molecules encoding antibody heavy and light chains (orfragments thereof), including such nucleic acid molecule(s) in a singlevector or separate vectors, and such nucleic acid molecule(s) present atone or more locations in a host cell.

The term “HER2,” as used herein, refers to any native, mature HER2 whichresults from processing of a HER2 precursor protein in a cell. The termincludes HER2 from any vertebrate source, including mammals such asprimates (e.g. humans and cynomolgus monkeys) and rodents (e.g., miceand rats), unless otherwise indicated. The term also includes naturallyoccurring variants of HER2, e.g., splice variants or allelic variants.The amino acid sequence of an exemplary human HER2 precursor protein,with signal sequence (with signal sequence, amino acids 1-22) is shownin SEQ ID NO: 1. The amino acid sequence of an exemplary mature humanHER2 is amino acids 23-1255 of SEQ ID NO: 1.

The term “HER2-positive cell” refers to a cell that expresses HER2 onits surface.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

A “naked antibody” refers to an antibody that is not conjugated to aheterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The nakedantibody may be present in a pharmaceutical formulation.

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150,000 daltons, composed oftwo identical light chains and two identical heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3).Similarly, from N- to C-terminus, each light chain has a variable region(VL), also called a variable light domain or a light chain variabledomain, followed by a constant light (CL) domain. The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain.

A “vial” is a container suitable for holding a liquid or lyophilizedpreparation. In one embodiment, the vial is a single-use vial, e.g. a20-cc single-use vial with a stopper.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:

-   -   100 times the fraction X/Y        where X is the number of amino acid residues scored as identical        matches by the sequence alignment program ALIGN-2 in that        program's alignment of A and B, and where Y is the total number        of amino acid residues in B. It will be appreciated that where        the length of amino acid sequence A is not equal to the length        of amino acid sequence B, the % amino acid sequence identity of        A to B will not equal the % amino acid sequence identity of B        to A. Unless specifically stated otherwise, all % amino acid        sequence identity values used herein are obtained as described        in the immediately preceding paragraph using the ALIGN-2        computer program.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology.

Desirable effects of treatment include, but are not limited to,preventing occurrence or recurrence of disease, alleviation of symptoms,diminishment of any direct or indirect pathological consequences of thedisease, preventing metastasis, decreasing the rate of diseaseprogression, amelioration or palliation of the disease state, andremission or improved prognosis. In some embodiments, antibodies of theinvention are used to delay development of a disease or to slow theprogression of a disease.

By “co-administering” is meant intravenously administering two (or more)drugs during the same administration, rather than sequential infusionsof the two or more drugs. Generally, this will involve combining the two(or more) drugs into the same IV bag prior to co-administration thereof.

A drug that is administered “concurrently” with one or more other drugsis administered during the same treatment cycle, on the same day oftreatment as the one or more other drugs, and, optionally, at the sametime as the one or more other drugs. For instance, for cancer therapiesgiven every 3 weeks, the concurrently administered drugs are eachadministered on day-1 of a 3-week cycle.

A “chemotherapy” is use of a chemotherapeutic agent useful in thetreatment of cancer.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer, regardless of mechanism of action. Classes ofchemotherapeutic agents include, but are not limited to: alkylatingagents, antimetabolites, spindle poison plant alkaloids,cytotoxic/antitumor antibiotics, topoisomerase inhibitors, antibodies,photosensitizers, and kinase inhibitors. Examples of chemotherapeuticagents include: anthracyclines, such as epirubicin or doxorubicin(ADRIAMYCIN®), cyclophosphamide (CYTOXAN®, NEOSAR®), anthracycline andcyclophosphamide in combination (“AC”); a taxane, e.g., docetaxel(TAXOTERE®) or paclitaxel (TAXOL®), 5-FU (fluorouracil, 5-fluorouracil,CAS No. 51-21-8), lapatinib (TYKERB®), capecitabine (XELODA®),gemcitabine (GEMZAR®, Lilly), PD-0325901 (CAS No. 391210-10-9, Pfizer),cisplatin (cis-diamine,dichloroplatinum(II), CAS No. 15663-27-1),carboplatin (CAS No. 41575-94-4), temozolomide(4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo [4.3.0]nona-2,7,9-triene-9-carboxamide, CAS No. 85622-93-1, TEMODAR®, TEMODAL®,Schering Plough), tamoxifen((Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethyl-ethanamine,NOLVADEX®, ISTUBAL®, VALODEX®).

More examples of chemotherapeutic agents include: oxaliplatin(ELOXATIN®, Sanofi), bortezomib (VELCADE®, Millennium Pharm.), sutent(SUNITINIB®, SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinibmesylate (GLEEVEC®, Novartis), XL-518 (MEK inhibitor, Exelixis, WO2007/044515), ARRY-886 (Mek inhibitor, AZD6244, Array BioPharma, AstraZeneca), SF-1126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235(PI3K inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis), PTK787/ZK222584 (Novartis), fulvestrant (FASLODEX®, AstraZeneca), leucovorin(folinic acid), rapamycin (sirolimus, RAPAMUNE®, Wyeth), lonafarnib(SARASAR™, SCH 66336, Schering Plough), sorafenib (NEXAVAR®, BAY43-9006,Bayer Labs), gefitinib (IRESSA®, AstraZeneca), irinotecan (CAMPTOSAR®,CPT-11, Pfizer), tipifarnib (ZARNESTRA™, Johnson & Johnson), ABRAXANE™(Cremophor-free), albumin-engineered nanoparticle formulations ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.),vandetanib (rINN, ZD6474, ZACTIMA®, AstraZeneca), chloranmbucil, AG1478,AG1571 (SU 5271; Sugen), temsirolimus (TORISEL®, Wyeth), pazopanib(GlaxoSmithKline), canfosfamide (TELCYTA®, Telik), thiotepa andcyclosphosphamide (CYTOXAN®, NEOSAR®); alkyl sulfonates such asbusulfan, improsulfan and piposulfan; aziridines such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelaminesincluding altretamine, triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analog topotecan); bryostatin; callystatin; CC-1065 (includingits adozelesin, carzelesin and bizelesin synthetic analogs);cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogs, KW-2189 andCB1-TM 1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,chlorophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, calicheamicin gammalI, calicheamicin omegaIl (Angew Chem.Intl. Ed. Engl. (1994) 33:183-186); dynemicin, dynemicin A;bisphosphonates, such as clodronate; an esperamicin; as well asneocarzinostatin chromophore and related chromoprotein enediyneantibiotic chromophores), aclacinomysins, actinomycin, authramycin,azaserine, bleomycins, cactinomycin, carabicin, carminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, porfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogs such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (T-2 toxin, verracurin A,roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine;mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (Ara-C);cyclophosphamide; thiotepa; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine(NAVELBINE®); novantrone; teniposide; edatrexate; daunomycin;aminopterin; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoids such as retinoic acid; andpharmaceutically acceptable salts, acids and derivatives of any of theabove.

A “fixed” or “flat” dose of a therapeutic agent herein refers to a dosethat is administered to a human patient without regard for the weight(WT) or body surface area (BSA) of the patient. The fixed or flat doseis therefore not provided as a mg/kg dose or a mg/m2 dose, but rather asan absolute amount of the therapeutic agent.

A “loading” dose herein generally comprises an initial dose of atherapeutic agent administered to a patient, and is followed by one ormore maintenance dose(s) thereof. Generally, a single loading dose isadministered, but multiple loading doses are contemplated herein.Usually, the amount of loading dose(s) administered exceeds the amountof the maintenance dose(s) administered and/or the loading dose(s) areadministered more frequently than the maintenance dose(s), so as toachieve the desired steady-state concentration of the therapeutic agentearlier than can be achieved with the maintenance dose(s).

A “maintenance” dose herein refers to one or more doses of a therapeuticagent administered to the patient over a treatment period. Usually, themaintenance doses are administered at spaced treatment intervals, suchas approximately every week, approximately every 2 weeks, approximatelyevery 3 weeks, or approximately every 4 weeks, preferably every 3 weeks.

“Infusion” or “infusing” refers to the introduction of a drug-containingsolution into the body through a vein for therapeutic purposes.Generally, this is achieved via an intravenous (IV) bag.

An “intravenous bag” or “IV bag” is a bag that can hold a solution whichcan be administered via the vein of a patient. In one embodiment, thesolution is a saline solution (e.g. about 0.9% or about 0.45% NaCl).Optionally, the IV bag is formed from polyolefin or polyvinal chloride.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindtet al. Kuby Immunology, 6^(th) ed., W.H. Freeman and Co., page 91(2007).) A single VH or VL domain may be sufficient to conferantigen-binding specificity. Furthermore, antibodies that bind aparticular antigen may be isolated using a VH or VL domain from anantibody that binds the antigen to screen a library of complementary VLor VH domains, respectively. See, e.g., Portolano et al., J. Immunol.150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors.”

“Alkyl” is C₁-C₁₈ hydrocarbon containing normal, secondary, tertiary orcyclic carbon atoms. Examples are methyl (Me, —CH₃), ethyl (Et,—CH₂CH₃), 1-propyl (n-Pr, n-propyl, —CH₂CH₂CH₃), 2-propyl (i-Pr,i-propyl, —CH(CH₃)₂), 1-butyl (n-Bu, n-butyl, —CH₂CH₂CH₂CH₃),2-methyl-1-propyl (i-Bu, i-butyl, —CH₂CH(CH₃)₂), 2-butyl (s-Bu, s-butyl,—CH(CH₃)CH₂CH₃), 2-methyl-2-propyl (t-Bu, t-butyl, —C(CH₃)₃), 1-pentyl(n-pentyl, —CH₂CH₂CH₂CH₂CH₃), 2-pentyl (—CH(CH₃)CH₂CH₂CH₃), 3-pentyl(—CH(CH₂CH₃)₂), 2-methyl-2-butyl (—C(CH₃)₂CH₂CH₃), 3-methyl-2-butyl(—CH(CH₃)CH(CH₃)₂), 3-methyl-1-butyl (—CH₂CH₂CH(CH₃)₂), 2-methyl-1-butyl(—CH₂CH(CH₃)CH₂CH₃), 1-hexyl (—CH₂CH₂CH₂CH₂CH₂CH₃), 2-hexyl(—CH(CH₃)CH₂CH₂CH₂CH₃), 3-hexyl (—CH(CH₂CH₃)(CH₂CH₂CH₃)),2-methyl-2-pentyl (—C(CH₃)₂CH₂CH₂CH₃), 3-methyl-2-pentyl(—CH(CH₃)CH(CH₃)CH₂CH₃), 4-methyl-2-pentyl (—CH(CH₃)CH₂CH(CH₃)₂),3-methyl-3-pentyl (—C(CH₃)(CH₂CH₃)₂), 2-methyl-3-pentyl(—CH(CH₂CH₃)CH(CH₃)₂), 2,3-dimethyl-2-butyl (—C(CH₃)₂CH(CH₃)₂),3,3-dimethyl-2-butyl (—CH(CH₃)C(CH₃)₃.

The term “C₁-C₈ alkyl,” as used herein refers to a straight chain orbranched, saturated or unsaturated hydrocarbon having from 1 to 8 carbonatoms. Representative “C₁-C₈ alkyl” groups include, but are not limitedto, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -n-hexyl,-n-heptyl, -n-octyl, -n-nonyl and -n-decyl; while branched C₁-C₈ alkylsinclude, but are not limited to, -isopropyl, -sec-butyl, -isobutyl,-tert-butyl, -isopentyl, 2-methylbutyl, unsaturated C₁-C₈ alkylsinclude, but are not limited to, -vinyl, -allyl, -1-butenyl, -2-butenyl,-isobutylenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl,-2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, 1-hexyl, 2-hexyl, 3-hexyl,-acetylenyl, -propynyl, -1-butynyl, -2-butynyl, -1-pentynyl,-2-pentynyl, -3-methyl-1 butynyl. A C₁-C₈ alkyl group can beunsubstituted or substituted with one or more groups including, but notlimited to, —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′,—C(O)OR′, —C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —SO₃R′, —S(O)₂R′,—S(O)R′, —OH, -halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN; where eachR′ is independently selected from H, —C₁-C₈ alkyl and aryl.

The term “C₁-C₁₂ alkyl,” as used herein refers to a straight chain orbranched, saturated or unsaturated hydrocarbon having from 1 to 12carbon atoms. A C₁-C₁₂ alkyl group can be unsubstituted or substitutedwith one or more groups including, but not limited to, —C₁-C₈ alkyl,—O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂,—C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —SO₃R′, —S(O)₂R′, —S(O)R′, —OH,-halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN; where each R′ isindependently selected from H, —C₁-C₈ alkyl and aryl.

The term “C₁-C₆ alkyl,” as used herein refers to a straight chain orbranched, saturated or unsaturated hydrocarbon having from 1 to 6 carbonatoms. Representative “C₁-C₆ alkyl” groups include, but are not limitedto, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -and n-hexyl; whilebranched C₁-C₆ alkyls include, but are not limited to, -isopropyl,-sec-butyl, -isobutyl, -tert-butyl, -isopentyl, and 2-methylbutyl;unsaturated C₁-C₆ alkyls include, but are not limited to, -vinyl,-allyl, -1-butenyl, -2-butenyl, and -isobutylenyl, -1-pentenyl,-2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl,-2,3-dimethyl-2-butenyl, 1-hexyl, 2-hexyl, and 3-hexyl. A C₁-C₆ alkylgroup can be unsubstituted or substituted with one or more groups, asdescribed above for C₁-C₈ alkyl group.

The term “C₁-C₄ alkyl,” as used herein refers to a straight chain orbranched, saturated or unsaturated hydrocarbon having from 1 to 4 carbonatoms. Representative “C₁-C₄ alkyl” groups include, but are not limitedto, -methyl, -ethyl, -n-propyl, -n-butyl; while branched C₁-C₄ alkylsinclude, but are not limited to, -isopropyl, -sec-butyl, -isobutyl,-tert-butyl; unsaturated C₁-C₄ alkyls include, but are not limited to,-vinyl, -allyl, -1-butenyl, -2-butenyl, and -isobutylenyl. A C₁-C₄ alkylgroup can be unsubstituted or substituted with one or more groups, asdescribed above for C₁-C₈ alkyl group.

“Alkoxy” is an alkyl group singly bonded to an oxygen. Exemplary alkoxygroups include, but are not limited to, methoxy (—OCH₃) and ethoxy(—OCH₂CH₃). A “C₁-C₈ alkoxy” is an alkoxy group with 1 to 5 carbonatoms. Alkoxy groups may can be unsubstituted or substituted with one ormore groups, as described above for alkyl groups.

“Alkenyl” is C₂-C₁₈ hydrocarbon containing normal, secondary, tertiaryor cyclic carbon atoms with at least one site of unsaturation, i.e. acarbon-carbon, sp² double bond. Examples include, but are not limitedto: ethylene or vinyl (—CH═CH₂), allyl (—CH₂CH═CH₂), cyclopentenyl(—C₅H7), and 5-hexenyl (—CH₂ CH₂CH₂CH₂CH═CH₂). A “C₂-C₈ alkenyl” is ahydrocarbon containing 2 to 8 normal, secondary, tertiary or cycliccarbon atoms with at least one site of unsaturation, i.e. acarbon-carbon, sp² double bond.

“Alkynyl” is C₂-C₁₈ hydrocarbon containing normal, secondary, tertiaryor cyclic carbon atoms with at least one site of unsaturation, i.e. acarbon-carbon, sp triple bond. Examples include, but are not limited to:acetylenic (—C═CH) and propargyl (—CH₂C═CH). A “C₂-C₈ alkynyl” is ahydrocarbon containing 2 to 8 normal, secondary, tertiary or cycliccarbon atoms with at least one site of unsaturation, i.e. acarbon-carbon, sp triple bond.

“Alkylene” refers to a saturated, branched or straight chain or cyclichydrocarbon radical of 1-18 carbon atoms, and having two monovalentradical centers derived by the removal of two hydrogen atoms from thesame or two different carbon atoms of a parent alkane. Typical alkyleneradicals include, but are not limited to: methylene (—CH₂—) 1,2-ethyl(—CH₂CH₂—), 1,3-propyl (—CH₂CH₂CH₂—), 1,4-butyl (—CH₂CH₂CH₂CH₂—), andthe like.

“Alkenylene” refers to an unsaturated, branched or straight chain orcyclic hydrocarbon radical of 2-18 carbon atoms, and having twomonovalent radical centers derived by the removal of two hydrogen atomsfrom the same or two different carbon atoms of a parent alkene. Typicalalkenylene radicals include, but are not limited to: 1,2-ethylene(—CH═CH—).

“Alkynylene” refers to an unsaturated, branched or straight chain orcyclic hydrocarbon radical of 2-18 carbon atoms, and having twomonovalent radical centers derived by the removal of two hydrogen atomsfrom the same or two different carbon atoms of a parent alkyne. Typicalalkynylene radicals include, but are not limited to: acetylene (—C≡C—),propargyl (—CH₂C═C—), and 4-pentynyl (—CH₂CH₂CH₂C≡C—).

“Aryl” refers to a carbocyclic aromatic group. Examples of aryl groupsinclude, but are not limited to, phenyl, naphthyl and anthracenyl. Acarbocyclic aromatic group or a heterocyclic aromatic group can beunsubstituted or substituted with one or more groups including, but notlimited to, —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′,—C(O)OR′, —C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′,—OH, -halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN; wherein each R′ isindependently selected from H, —C₁-C₈ alkyl and aryl.

A “C₅-C₂₀ aryl” is an aryl group with 5 to 20 carbon atoms in thecarbocyclic aromatic rings. Examples of C₅-C₂₀ aryl groups include, butare not limited to, phenyl, naphthyl and anthracenyl. A C₅-C₂₀ arylgroup can be substituted or unsubstituted as described above for arylgroups. A “C₅-C₁₄ aryl” is an aryl group with 5 to 14 carbon atoms inthe carbocyclic aromatic rings. Examples of C₅-C₁₄ aryl groups include,but are not limited to, phenyl, naphthyl and anthracenyl. A C₅-C₁₄ arylgroup can be substituted or unsubstituted as described above for arylgroups.

An “arylene” is an aryl group which has two covalent bonds and can be inthe ortho, meta, or para configurations as shown in the followingstructures:

in which the phenyl group can be unsubstituted or substituted with up tofour groups including, but not limited to, —C₁-C₈ alkyl, —O—(C₁-C₈alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′,—C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, -halogen, —N₃, —NH₂,—NH(R′), —N(R′)₂ and —CN; wherein each R′ is independently selected fromH, —C₁-C₈ alkyl and aryl.

“Arylalkyl” refers to an acyclic alkyl radical in which one of thehydrogen atoms bonded to a carbon atom, typically a terminal or sp³carbon atom, is replaced with an aryl radical. Typical arylalkyl groupsinclude, but are not limited to, benzyl, 2-phenylethan-1-yl,2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl,2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl and thelike. The arylalkyl group comprises 6 to 20 carbon atoms, e.g. the alkylmoiety, including alkanyl, alkenyl or alkynyl groups, of the arylalkylgroup is 1 to 6 carbon atoms and the aryl moiety is 5 to 14 carbonatoms.

“Heteroarylalkyl” refers to an acyclic alkyl radical in which one of thehydrogen atoms bonded to a carbon atom, typically a terminal or sp³carbon atom, is replaced with a heteroaryl radical. Typicalheteroarylalkyl groups include, but are not limited to,2-benzimidazolylmethyl, 2-furylethyl, and the like. The heteroarylalkylgroup comprises 6 to 20 carbon atoms, e.g. the alkyl moiety, includingalkanyl, alkenyl or alkynyl groups, of the heteroarylalkyl group is 1 to6 carbon atoms and the heteroaryl moiety is 5 to 14 carbon atoms and 1to 3 heteroatoms selected from N, O, P, and S. The heteroaryl moiety ofthe heteroarylalkyl group may be a monocycle having 3 to 7 ring members(2 to 6 carbon atoms or a bicycle having 7 to 10 ring members (4 to 9carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S), forexample: a bicyclo [4,5], [5,5], [5,6], or [6,6] system.

“Substituted alkyl,” “substituted aryl,” and “substituted arylalkyl”mean alkyl, aryl, and arylalkyl respectively, in which one or morehydrogen atoms are each independently replaced with a substituent.Typical substituents include, but are not limited to, —X, —R, —O—, —OR,—SR, —S⁻, —NR₂, —NR₃, ═NR, —CX₃, —CN, —OCN, —SCN, —N═C═O, —NCS, —NO,—NO₂, ═N₂, —N₃, NC(═O)R, —C(═O)R, —C(═O)NR₂, —SO₃ ⁻, —SO₃H, —S(═O)₂R,—OS(═O)₂OR, —S(═O)₂NR, —S(═O)R, —OP(═O)(OR)₂, —P(═O)(OR)₂, —PO⁻ ₃,—PO₃H₂, —C(═O)R, —C(═O)X, —C(═S)R, —CO₂R, —CO₂ ⁻, —C(═S)OR, —C(═O)SR,—C(═S)SR, —C(═O)NR₂, —C(═S)NR₂, —C(═NR)NR₂, where each X isindependently a halogen: F, Cl, Br, or I; and each R is independently—H, C₂-C₁₈ alkyl, C₆-C₂₀ aryl, C₃-C₁₄ heterocycle, protecting group orprodrug moiety. Alkylene, alkenylene, and alkynylene groups as describedabove may also be similarly substituted.

“Heteroaryl” and “heterocycle” refer to a ring system in which one ormore ring atoms is a heteroatom, e.g. nitrogen, oxygen, and sulfur. Theheterocycle radical comprises 3 to 20 carbon atoms and 1 to 3heteroatoms selected from N, O, P, and S. A heterocycle may be amonocycle having 3 to 7 ring members (2 to 6 carbon atoms and 1 to 3heteroatoms selected from N, O, P, and S) or a bicycle having 7 to 10ring members (4 to 9 carbon atoms and 1 to 3 heteroatoms selected fromN, O, P, and S), for example: a bicyclo [4,5], [5,5], [5,6], or [6,6]system.

Exemplary heterocycles are described, e.g., in Paquette, Leo A.,“Principles of Modern Heterocyclic Chemistry” (W. A. Benjamin, New York,1968), particularly Chapters 1, 3, 4, 6, 7, and 9; “The Chemistry ofHeterocyclic Compounds, A series of Monographs” (John Wiley & Sons, NewYork, 1950 to present), in particular Volumes 13, 14, 16, 19, and 28;and J. Am. Chem. Soc. (1960) 82:5566.

Examples of heterocycles include by way of example and not limitationpyridyl, dihydroypyridyl, tetrahydropyridyl (piperidyl), thiazolyl,tetrahydrothiophenyl, sulfur oxidized tetrahydrothiophenyl, pyrimidinyl,furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl,benzofuranyl, thianaphthalenyl, indolyl, indolenyl, quinolinyl,isoquinolinyl, benzimidazolyl, piperidinyl, 4-piperidonyl, pyrrolidinyl,2-pyrrolidonyl, pyrrolinyl, tetrahydrofuranyl, bis-tetrahydrofuranyl,tetrahydropyranyl, bis-tetrahydropyranyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl,azocinyl, triazinyl, 6H-1,2,5-thiadiazinyl, 2H,6H-1,5,2-dithiazinyl,thienyl, thianthrenyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl,phenoxathinyl, 2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl,pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl,4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl,quinazolinyl, cinnolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl,β-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, isochromanyl,chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl,piperazinyl, indolinyl, isoindolinyl, quinuclidinyl, morpholinyl,oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl,and isatinoyl.

By way of example and not limitation, carbon bonded heterocycles arebonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5, or6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position 2,3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan,tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole,position 2, 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4,or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of anaziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6,7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of anisoquinoline. Still more typically, carbon bonded heterocycles include2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl,4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl,4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl,5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or 5-thiazolyl.

By way of example and not limitation, nitrogen bonded heterocycles arebonded at position 1 of an aziridine, azetidine, pyrrole, pyrrolidine,2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline,3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline,piperidine, piperazine, indole, indoline, 1H-indazole, position 2 of aisoindole, or isoindoline, position 4 of a morpholine, and position 9 ofa carbazole, or β-carboline. Still more typically, nitrogen bondedheterocycles include 1-aziridyl, 1-azetedyl, 1-pyrrolyl, 1-imidazolyl,1-pyrazolyl, and 1-piperidinyl.

A “C₃-C₈ heterocycle” refers to an aromatic or non-aromatic C₃-C₈carbocycle in which one to four of the ring carbon atoms areindependently replaced with a heteroatom from the group consisting of O,S and N. Representative examples of a C₃-C₈ heterocycle include, but arenot limited to, benzofuranyl, benzothiophene, indolyl, benzopyrazolyl,coumarinyl, isoquinolinyl, pyrrolyl, thiophenyl, furanyl, thiazolyl,imidazolyl, pyrazolyl, triazolyl, quinolinyl, pyrimidinyl, pyridinyl,pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl, isoxazolyl andtetrazolyl. A C₃-C₈ heterocycle can be unsubstituted or substituted withup to seven groups including, but not limited to, —C₁-C₈ alkyl,—O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂,—C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, -halogen, —N₃,—NH₂, —NH(R′), —N(R′)₂ and —CN; wherein each R′ is independentlyselected from H, —C₁-C₈ alkyl and aryl.

“C₃-C₈ heterocyclo” refers to a C₃-C₈ heterocycle group defined abovewherein one of the heterocycle group's hydrogen atoms is replaced with abond. A C₃-C₈ heterocyclo can be unsubstituted or substituted with up tosix groups including, but not limited to, —C₁-C₈ alkyl, —O—(C₁-C₅alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′,—C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, -halogen, —N₃, —NH₂,—NH(R′), —N(R′)₂ and —CN; wherein each R′ is independently selected fromH, —C₁-C₈ alkyl and aryl.

A “C₃-C₂₀ heterocycle” refers to an aromatic or non-aromatic C₃-C₈carbocycle in which one to four of the ring carbon atoms areindependently replaced with a heteroatom from the group consisting of O,S and N. A C₃-C₂₀ heterocycle can be unsubstituted or substituted withup to seven groups including, but not limited to, —C₁-C₈ alkyl,—O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂,—C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, -halogen, —N₃,—NH₂, —NH(R′), —N(R′)₂ and —CN; wherein each R′ is independentlyselected from H, —C₁-C₈ alkyl and aryl.

“C₃-C₂₀ heterocyclo” refers to a C₃-C₂₀ heterocycle group defined abovewherein one of the heterocycle group's hydrogen atoms is replaced with abond.

“Carbocycle” means a saturated or unsaturated ring having 3 to 7 carbonatoms as a monocycle or 7 to 12 carbon atoms as a bicycle. Monocycliccarbocycles have 3 to 6 ring atoms, still more typically 5 or 6 ringatoms. Bicyclic carbocycles have 7 to 12 ring atoms, e.g. arranged as abicyclo [4,5], [5,5], [5,6] or [6,6] system, or 9 or 10 ring atomsarranged as a bicyclo [5,6] or [6,6] system. Examples of monocycliccarbocycles include cyclopropyl, cyclobutyl, cyclopentyl,1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl,1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cycloheptyl,and cyclooctyl.

A “C₃-C₈ carbocycle” is a 3-, 4-, 5-, 6-, 7- or 8-membered saturated orunsaturated non-aromatic carbocyclic ring. Representative C₃-C₈carbocycles include, but are not limited to, -cyclopropyl, -cyclobutyl,-cyclopentyl, -cyclopentadienyl, -cyclohexyl, -cyclohexenyl,-1,3-cyclohexadienyl, -1,4-cyclohexadienyl, -cycloheptyl,-1,3-cycloheptadienyl, -1,3,5-cycloheptatrienyl, -cyclooctyl, and-cyclooctadienyl. A C₃-C₈ carbocycle group can be unsubstituted orsubstituted with one or more groups including, but not limited to,—C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′,—C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH,-halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN; where each R′ isindependently selected from H, —C₁-C₈ alkyl and aryl.

A “C₃-C₈ carbocyclo” refers to a C₃-C₈ carbocycle group defined abovewherein one of the carbocycle groups' hydrogen atoms is replaced with abond.

“Linker” refers to a chemical moiety comprising a covalent bond or achain of atoms that covalently attaches an antibody to a drug moiety. Invarious embodiments, linkers include a divalent radical such as analkyldiyl, an aryldiyl, a heteroaryldiyl, moieties such as:—(CR₂)_(n)O(CR₂)_(n)—, repeating units of alkyloxy (e.g. polyethylenoxy,PEG, polymethyleneoxy) and alkylamino (e.g. polyethyleneamino,Jeffamine™); and diacid ester and amides including succinate,succinamide, diglycolate, malonate, and caproamide. In variousembodiments, linkers can comprise one or more amino acid residues, suchas valine, phenylalanine, lysine, and homolysine.

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “stereoisomers” refers to compounds which have identicalchemical constitution, but differ with regard to the arrangement of theatoms or groups in space.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g. melting points,boiling points, spectral properties, and reactivities. Mixtures ofdiastereomers may separate under high resolution analytical proceduressuch as electrophoresis and chromatography.

“Enantiomers” refer to two stereoisomers of a compound which arenon-superimposable mirror images of one another.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., NewYork. Many organic compounds exist in optically active forms, i.e., theyhave the ability to rotate the plane of plane-polarized light. Indescribing an optically active compound, the prefixes D and L, or R andS, are used to denote the absolute configuration of the molecule aboutits chiral center(s). The prefixes d and 1 or (+) and (−) are employedto designate the sign of rotation of plane-polarized light by thecompound, with (−) or 1 meaning that the compound is levorotatory. Acompound prefixed with (+) or d is dextrorotatory. For a given chemicalstructure, these stereoisomers are identical except that they are mirrorimages of one another. A specific stereoisomer may also be referred toas an enantiomer, and a mixture of such isomers is often called anenantiomeric mixture. A 50:50 mixture of enantiomers is referred to as aracemic mixture or a racemate, which may occur where there has been nostereoselection or stereospecificity in a chemical reaction or process.The terms “racemic mixture” and “racemate” refer to an equimolar mixtureof two enantiomeric species, devoid of optical activity.

“Leaving group” refers to a functional group that can be substituted byanother functional group. Certain leaving groups are well known in theart, and examples include, but are not limited to, a halide (e.g.,chloride, bromide, iodide), methanesulfonyl (mesyl), p-toluenesulfonyl(tosyl), trifluoromethylsulfonyl (triflate), andtrifluoromethylsulfonate.

The term “protecting group” refers to a substituent that is commonlyemployed to block or protect a particular functionality while reactingother functional groups on the compound. For example, an“amino-protecting group” is a substituent attached to an amino groupthat blocks or protects the amino functionality in the compound.Suitable amino-protecting groups include, but are not limited to,acetyl, trifluoroacetyl, t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ)and 9-fluorenylmethylenoxycarbonyl (Fmoc). For a general description ofprotecting groups and their use, see T. W. Greene, Protective Groups inOrganic Synthesis, John Wiley & Sons, New York, 1991, or a lateredition.

II. COMPOSITIONS AND METHODS

In one aspect, the invention is based, in part, on antibodies that bindto HER2 and immunoconjugates comprising such antibodies. Antibodies andimmunoconjugates of the invention are useful, e.g., for the diagnosis ortreatment of HER2-positive cancers.

A. Exemplary Anti-HER2 Antibodies

Provided herein are isolated antibodies that bind to domain I of HER2.In some embodiments, the antibodies do not interfere with trastuzumaband/or pertuzumab binding to HER2.

In some embodiments, the antibodies do not interfere with trastuzumabbinding to HER2 and do not interfere with pertuzumab binding to HER2. Inany of the embodiments described herein, the antibodies may bemonoclonal antibodies. In some embodiments, the antibodies may be humanantibodies, humanized antibodies, or chimeric antibodies.

An exemplary naturally occurring human HER2 precursor protein sequence,with signal sequence (amino acids 1-22) is provided in SEQ ID NO: 1, andthe corresponding mature HER2 protein sequence corresponds to aminoacids 23-1255 of SEQ ID NO: 1. In some embodiments, domain I of HER2 hasthe amino acid sequence of SEQ ID NO: 35, domain II has the amino acidsequence of SEQ ID NO: 36, domain III has the amino acid sequence of SEQID NO: 37, and domain IV has the amino acid sequence of SEQ ID NO: 38(see FIG. 10).

Antibody hu7C2 and Other Embodiments

In some embodiments, an anti-HER2 antibody is provided that comprises atleast one, two, three, four, five, or six HVRs selected from (a) HVR-H1comprising the amino acid sequence of SEQ ID NO: 15; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 16; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 17; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO: 12; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 13; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO: 14. In someembodiments, an anti-HER2 antibody is provided that comprises an HVR-H2comprising the amino acid sequence of SEQ ID NO: 16 and at least one,two, three, four, or five HVRs selected from (a) HVR-H1 comprising theamino acid sequence of SEQ ID NO: 15; (b) HVR-H3 comprising the aminoacid sequence of SEQ ID NO: 17; (c) HVR-L1 comprising the amino acidsequence of SEQ ID NO: 12; (d) HVR-L2 comprising the amino acid sequenceof SEQ ID NO: 13; and (e) HVR-L3 comprising the amino acid sequence ofSEQ ID NO: 14.

In one aspect, an antibody is provided that comprises at least one, atleast two, or all three VH HVR sequences selected from (a) HVR-H 1comprising the amino acid sequence of SEQ ID NO: 15; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 16; and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 17. In one embodiment,the antibody comprises HVR-H3 comprising the amino acid sequence of SEQID NO: 17. In one embodiment, the antibody comprises HVR-H2 comprisingthe amino acid sequence of SEQ ID NO: 16. In another embodiment, theantibody comprises HVR-H3 comprising the amino acid sequence of SEQ IDNO: 17 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14.In a further embodiment, the antibody comprises HVR-H3 comprising theamino acid sequence of SEQ ID NO: 17, HVR-L3 comprising the amino acidsequence of SEQ ID NO: 14, and HVR-H2 comprising the amino acid sequenceof SEQ ID NO: 16. In a further embodiment, the antibody comprises (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO: 15; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 16; and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 17.

In another aspect, an antibody is provided that comprises at least one,at least two, or all three VL HVR sequences selected from (a) HVR-L 1comprising the amino acid sequence of SEQ ID NO: 12; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO: 13; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO: 14. In one embodiment,the antibody comprises (a) HVR-L1 comprising the amino acid sequence ofSEQ ID NO: 12; (b) HVR-L2 comprising the amino acid sequence of SEQ IDNO: 13; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:14.

In another aspect, an antibody comprises (a) a VH domain comprising atleast one, at least two, or all three VH HVR sequences selected from (i)HVR-H 1 comprising the amino acid sequence of SEQ ID NO: 15, (ii) HVR-H2comprising the amino acid sequence of SEQ ID NO: 16, and (iii) HVR-H3comprising an amino acid sequence selected from SEQ ID NO: 17; and (b) aVL domain comprising at least one, at least two, or all three VL HVRsequences selected from (i) HVR-L1 comprising the amino acid sequence ofSEQ ID NO: 12, (ii) HVR-L2 comprising the amino acid sequence of SEQ IDNO: 13, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:14.

In another aspect, an antibody is provided that comprises (a) HVR-H1comprising the amino acid sequence of SEQ ID NO: 15; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 16; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 17; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO: 12; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 13; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO: 14.

In any of the above embodiments, an anti-HER2 antibody is humanized. Inone embodiment, an anti-HER2 antibody comprises HVRs as in any of theabove embodiments, and further comprises a human acceptor framework,e.g. a human immunoglobulin framework or a human consensus framework. Incertain embodiments, the human acceptor framework is the human VL kappaIV consensus (VL_(KIV)) framework and/or the VH framework VH_(I). Incertain embodiments, the human acceptor framework is the human VL kappaIV consensus (VL_(KIV)) framework and/or the VH framework VH₁ comprisingany one of the mutations described herein.

In another aspect, an anti-HER2 antibody comprises a heavy chainvariable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO: 11. In certain embodiments, a VH sequence havingat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity tothe amino acid sequence of SEQ ID NO: 11 contains substitutions (e.g.,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-HER2 antibody comprising that sequenceretains the ability to bind to HER2. In certain embodiments, a total of1 to 10 amino acids have been substituted, inserted and/or deleted inSEQ ID NO: 11. In certain embodiments, a total of 1 to 5 amino acidshave been substituted, inserted and/or deleted in SEQ ID NO: 11. Incertain embodiments, substitutions, insertions, or deletions occur inregions outside the HVRs (i.e., in the FRs). Optionally, the anti-HER2antibody comprises the VH sequence of SEQ ID NO: 11, includingpost-translational modifications of that sequence. In a particularembodiment, the VH comprises one, two or three HVRs selected from: (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO: 15, (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 16, and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 17.

In another aspect, an anti-HER2 antibody is provided, wherein theantibody comprises a light chain variable domain (VL) having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 10. In certainembodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO:10 contains substitutions (e.g., conservative substitutions),insertions, or deletions relative to the reference sequence, but ananti-HER2 antibody comprising that sequence retains the ability to bindto HER2. In certain embodiments, a total of 1 to 10 amino acids havebeen substituted, inserted and/or deleted in SEQ ID NO: 10. In certainembodiments, a total of 1 to 5 amino acids have been substituted,inserted and/or deleted in SEQ ID NO: 10. In certain embodiments, thesubstitutions, insertions, or deletions occur in regions outside theHVRs (i.e., in the FRs). Optionally, the anti-HER2 antibody comprisesthe VL sequence of SEQ ID NO: 10, including post-translationalmodifications of that sequence. In a particular embodiment, the VLcomprises one, two or three HVRs selected from (a) HVR-L1 comprising theamino acid sequence of SEQ ID NO: 12; (b) HVR-L2 comprising the aminoacid sequence of SEQ ID NO: 13; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO: 14.

In another aspect, an anti-HER2 antibody is provided, wherein theantibody comprises a VH as in any of the embodiments provided above, anda VL as in any of the embodiments provided above.

In one embodiment, the antibody comprises the VH and VL sequences in SEQID NO: 11 and SEQ ID NO: 10, respectively, including post-translationalmodifications of those sequences.

In a further aspect, provided are herein are antibodies that bind to thesame epitope as an anti-HER2 antibody provided herein. For example, incertain embodiments, an antibody is provided that binds to the sameepitope as an anti-HER2 antibody comprising a VH sequence of SEQ ID NO:11 and a VL sequence of SEQ ID NO: 10, respectively.

In another aspect, an anti-HER2 antibody comprises a heavy chainsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:19. In certain embodiments, a heavy chain sequence having at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the aminoacid sequence of SEQ ID NO: 19 contains substitutions (e.g.,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-HER2 antibody comprising that sequenceretains the ability to bind to HER2. In certain embodiments, a total of1 to 10 amino acids have been substituted, inserted and/or deleted inSEQ ID NO: 19. In certain embodiments, a total of 1 to 5 amino acidshave been substituted, inserted and/or deleted in SEQ ID NO: 19. Incertain embodiments, substitutions, insertions, or deletions occur inregions outside the HVRs (i.e., in the FRs). Optionally, the anti-HER2antibody comprises the heavy chain sequence of SEQ ID NO: 19, includingpost-translational modifications of that sequence. In a particularembodiment, the heavy chain comprises one, two or three HVRs selectedfrom: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 15,(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 16, and (c)HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17.

In another aspect, an anti-HER2 antibody is provided, wherein theantibody comprises a light chain having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the aminoacid sequence of SEQ ID NO: 18. In certain embodiments, a light chainsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity to the amino acid sequence of SEQ ID NO: 18 containssubstitutions (e.g., conservative substitutions), insertions, ordeletions relative to the reference sequence, but an anti-HER2 antibodycomprising that sequence retains the ability to bind to HER2. In certainembodiments, a total of 1 to 10 amino acids have been substituted,inserted and/or deleted in SEQ ID NO: 18. In certain embodiments, atotal of 1 to 5 amino acids have been substituted, inserted and/ordeleted in SEQ ID NO: 18. In certain embodiments, the substitutions,insertions, or deletions occur in regions outside the HVRs (i.e., in theFRs). Optionally, the anti-HER2 antibody comprises the light chainsequence of SEQ ID NO: 18, including post-translational modifications ofthat sequence. In a particular embodiment, the light chain comprisesone, two or three HVRs selected from (a) HVR-L 1 comprising the aminoacid sequence of SEQ ID NO: 12; (b) HVR-L2 comprising the amino acidsequence of SEQ ID NO: 13; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO: 14.

In another aspect, an anti-HER2 antibody is provided, wherein theantibody comprises a heavy chain as in any of the embodiments providedabove, and a light chain as in any of the embodiments provided above.

In one embodiment, the antibody comprises the heavy chain and lightchain sequences in SEQ ID NO: 19 and SEQ ID NO: 18, respectively,including post-translational modifications of those sequences.

In a further aspect, provided are herein are antibodies that bind to thesame epitope as an anti-HER2 antibody provided herein. For example, incertain embodiments, an antibody is provided that binds to the sameepitope as an anti-HER2 antibody comprising a heavy chain sequence ofSEQ ID NO: 19 and a light chain sequence of SEQ ID NO: 18, respectively.

Provided herein are antibodies comprising a light chain variable domaincomprising the HVR1-LC, HVR2-LC and HVR3-LC sequence according to Kabatnumbering as depicted in FIG. 1 and a heavy chain variable domaincomprising the HVR1-HC, HVR2-HC and HVR3-HC sequence according to Kabatnumbering as depicted in FIG. 2. In some embodiments, the antibodycomprises a light chain variable domain comprising the HVR1-LC, HVR2-LCand/or HVR3-LC sequence, and the FR1-LC, FR2-LC, FR3-LC and/or FR4-LCsequence as depicted in FIG. 1. In some embodiments, the antibodycomprises a heavy chain variable domain comprising the HVR1-HC, HVR2-HCand/or HVR3-HC sequence, and the FR1-HC, FR2-HC, FR3-HC and/or FR4-HCsequence as depicted in FIG. 2.

In a further aspects, an anti-HER2 antibody according to any of theabove embodiments is a monoclonal antibody, including a human antibody.In one embodiment, an anti-HER2 antibody is an antibody fragment, e.g.,a Fv, Fab, Fab′, scFv, diabody, or F(ab′)₂ fragment. In anotherembodiment, the antibody is a substantially full length antibody, e.g.,an IgG1 antibody, IgG2a antibody or other antibody class or isotype asdefined herein.

In a further aspect, an anti-HER2 antibody according to any of the aboveembodiments may incorporate any of the features, singly or incombination, as described below.

1. Antibody Affinity

In certain embodiments, an antibody provided herein has a dissociationconstant (Kd) of ≤1 μM, ≤100 nM, ≤50 nM, ≤10 nM, ≤5 nM, ≤1 nM, ≤0.1 nM,≤0.01 nM, or ≤0.001 nM, and optionally is ≥10⁻¹³ M. (e.g. 10⁻⁸ M orless, e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ M to 10⁻¹³ M).

In one embodiment, Kd is measured by a radiolabeled antigen bindingassay (RIA) performed with the Fab version of an antibody of interestand its antigen as described by the following assay. Solution bindingaffinity of Fabs for antigen is measured by equilibrating Fab with aminimal concentration of (¹²⁵I)-labeled antigen in the presence of atitration series of unlabeled antigen, then capturing bound antigen withan anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol.293:865-881(1999)). To establish conditions for the assay, MICROTITER®multi-well plates (Thermo Scientific) are coated overnight with 5 μg/mlof a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate(pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin inPBS for two to five hours at room temperature (approximately 23° C.). Ina non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [¹²⁵I]-antigen aremixed with serial dilutions of a Fab of interest (e.g., consistent withassessment of the anti-VEGF antibody, Fab-12, in Presta et al., CancerRes. 57:4593-4599 (1997)). The Fab of interest is then incubatedovernight; however, the incubation may continue for a longer period(e.g., about 65 hours) to ensure that equilibrium is reached.Thereafter, the mixtures are transferred to the capture plate forincubation at room temperature (e.g., for one hour). The solution isthen removed and the plate washed eight times with 0.1% polysorbate 20(TWEEN-20®) in PBS. When the plates have dried, 150 μl/well ofscintillant (MICROSCINT-20™; Packard) is added, and the plates arecounted on a TOPCOUNT™ gamma counter (Packard) for ten minutes.Concentrations of each Fab that give less than or equal to 20% ofmaximal binding are chosen for use in competitive binding assays.

According to another embodiment, Kd is measured using surface plasmonresonance assays using a BIACORE®-2000 or a BIACORE®-3000 (BIAcore,Inc., Piscataway, N.J.) at 25° C. with immobilized antigen CM5 chips at˜10 response units (RU). Briefly, carboxymethylated dextran biosensorchips (CM5, BIACORE, Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (˜0.2μM) before injection at a flow rate of 5 μl/minute to achieveapproximately 10 response units (RU) of coupled protein. Following theinjection of antigen, 1 M ethanolamine is injected to block unreactedgroups. For kinetics measurements, two-fold serial dilutions of Fab(0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20(TWEEN-20™) surfactant (PBST) at 25° C. at a flow rate of approximately25 μl/min. Association rates (k_(on)) and dissociation rates (k_(off))are calculated using a simple one-to-one Langmuir binding model(BIACORE® Evaluation Software version 3.2) by simultaneously fitting theassociation and dissociation sensorgrams. The equilibrium dissociationconstant (Kd) is calculated as the ratio k_(off)/k_(on). See, e.g., Chenet al., J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 10⁶ M⁻¹s⁻¹ by the surface plasmon resonance assay above, then the on-rate canbe determined by using a fluorescent quenching technique that measuresthe increase or decrease in fluorescence emission intensity(excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence ofincreasing concentrations of antigen as measured in a spectrometer, suchas a stop-flow equipped spectrophometer (Aviv Instruments) or a8000-series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with astirred cuvette.

2. Antibody Fragments

In certain embodiments, an antibody provided herein is an antibodyfragment. Antibody fragments include, but are not limited to, Fab, Fab′,Fab′-SH, F(ab′)₂, Fv, and scFv fragments, and other fragments describedbelow. For a review of certain antibody fragments, see Hudson et al.Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g.,Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315(1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and5,587,458. For discussion of Fab and F(ab′)₂ fragments comprisingsalvage receptor binding epitope residues and having increased in vivohalf-life, see U.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc.Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodiesare also described in Hudson et al., Nat. Med. 9:129-134 (2003).

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g. E. coli or phage), asdescribed herein.

3. Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimericantibody. Certain chimeric antibodies are described, e.g., in U.S. Pat.No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)). In one example, a chimeric antibody comprises anon-human variable region (e.g., a variable region derived from a mouse,rat, hamster, rabbit, or non-human primate, such as a monkey) and ahuman constant region. In a further example, a chimeric antibody is a“class switched” antibody in which the class or subclass has beenchanged from that of the parent antibody. Chimeric antibodies includeantigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, e.g., CDRs, (or portions thereof)are derived from a non-human antibody, and FRs (or portions thereof) arederived from human antibody sequences. A humanized antibody optionallywill also comprise at least a portion of a human constant region. Insome embodiments, some FR residues in a humanized antibody aresubstituted with corresponding residues from a non-human antibody (e.g.,the antibody from which the HVR residues are derived), e.g., to restoreor improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and arefurther described, e.g., in Riechmann et al., Nature 332:323-329 (1988);Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S.Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri etal., Methods 36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan,Mol. Immunol. 28:489-498 (1991) (describing “resurfacing”); Dall'Acquaet al., Methods 36:43-60 (2005) (describing “FR shuffling”); and Osbournet al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer,83:252-260 (2000) (describing the “guided selection” approach to FRshuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light or heavy chain variable regions (see, e.g.,Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta etal. J. Immunol., 151:2623 (1993)); human mature (somatically mutated)framework regions or human germline framework regions (see, e.g.,Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and frameworkregions derived from screening FR libraries (see, e.g., Baca et al., J.Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.271:22611-22618 (1996)).

4. Human Antibodies

In certain embodiments, an antibody provided herein is a human antibody.Human antibodies can be produced using various techniques known in theart. Human antibodies are described generally in van Dijk and van deWinkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin.Immunol. 20:450-459 (2008).

Human antibodies may be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Seealso, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™technology; U.S. Pat. No. 5,770,429 describing HUMAB® technology; U.S.Pat. No. 7,041,870 describing K-M MOUSE® technology, and U.S. PatentApplication Publication No. US 2007/0061900, describing VELOCIMOUSE®technology). Human variable regions from intact antibodies generated bysuch animals may be further modified, e.g., by combining with adifferent human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, e.g., Kozbor J.Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Humanantibodies generated via human B-cell hybridoma technology are alsodescribed in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562(2006). Additional methods include those described, for example, in U.S.Pat. No. 7,189,826 (describing production of monoclonal human IgMantibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue,26(4):265-268 (2006) (describing human-human hybridomas). Humanhybridoma technology (Trioma technology) is also described in Vollmersand Brandlein, Histology and Histopathology, 20(3):927-937 (2005) andVollmers and Brandlein, Methods and Findings in Experimental andClinical Pharmacology, 27(3): 185-91 (2005).

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described below.

5. Library-Derived Antibodies

Antibodies of the invention may be isolated by screening combinatoriallibraries for antibodies with the desired activity or activities. Forexample, a variety of methods are known in the art for generating phagedisplay libraries and screening such libraries for antibodies possessingthe desired binding characteristics. Such methods are reviewed, e.g., inHoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien etal., ed., Human Press, Totowa, N.J., 2001) and further described, e.g.,in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992);Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo,ed., Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol.338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472(2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004).

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al., Ann. Rev. Immunol.,12: 433-455 (1994). Phage typically display antibody fragments, eitheras single-chain Fv (scFv) fragments or as Fab fragments. Libraries fromimmunized sources provide high-affinity antibodies to the immunogenwithout the requirement of constructing hybridomas. Alternatively, thenaive repertoire can be cloned (e.g., from human) to provide a singlesource of antibodies to a wide range of non-self and also self antigenswithout any immunization as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be made syntheticallyby cloning unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro, as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patentpublications describing human antibody phage libraries include, forexample: U.S. Pat. No. 5,750,373, and US Patent Publication Nos.2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody librariesare considered human antibodies or human antibody fragments herein.

6. Multispecific Antibodies

In certain embodiments, an antibody provided herein is a multispecificantibody, e.g. a bispecific antibody. Multispecific antibodies aremonoclonal antibodies that have binding specificities for at least twodifferent sites. In certain embodiments, one of the bindingspecificities is for HER2 and the other is for any other antigen. Incertain embodiments, one of the binding specificities is for HER2 andthe other is for CD3. See, e.g., U.S. Pat. No. 5,821,337. In certainembodiments, bispecific antibodies may bind to two different epitopes ofHER2. Bispecific antibodies may also be used to localize cytotoxicagents to cells which express HER2. Bispecific antibodies can beprepared as full length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein andCuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al.,EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g.,U.S. Pat. No. 5,731,168). The term “knob-into-hole” or “KnH” technologyas used herein refers to the technology directing the pairing of twopolypeptides together in vitro or in vivo by introducing a protuberance(knob) into one polypeptide and a cavity (hole) into the otherpolypeptide at an interface in which they interact. For example, KnHshave been introduced in the Fc:Fc binding interfaces, CL:CH1 interfacesor VH/VL interfaces of antibodies (see, e.g., US 2011/0287009,US2007/0178552, WO 96/027011, WO 98/050431, Zhu et al., 1997, ProteinScience 6:781-788, and WO2012/106587). In some embodiments, KnHs drivethe pairing of two different heavy chains together during themanufacture of multispecific antibodies. For example, multispecificantibodies having KnH in their Fc regions can further comprise singlevariable domains linked to each Fc region, or further comprise differentheavy chain variable domains that pair with similar or different lightchain variable domains. KnH technology can be also be used to pair twodifferent receptor extracellular domains together or any otherpolypeptide sequences that comprises different target recognitionsequences (e.g., including affibodies, peptibodies and other Fcfusions).

The term “knob mutation” as used herein refers to a mutation thatintroduces a protuberance (knob) into a polypeptide at an interface inwhich the polypeptide interacts with another polypeptide. In someembodiments, the other polypeptide has a hole mutation.

The term “hole mutation” as used herein refers to a mutation thatintroduces a cavity (hole) into a polypeptide at an interface in whichthe polypeptide interacts with another polypeptide. In some embodiments,the other polypeptide has a knob mutation.

A brief nonlimiting discussion is provided below.

A “protuberance” refers to at least one amino acid side chain whichprojects from the interface of a first polypeptide and is thereforepositionable in a compensatory cavity in the adjacent interface (i.e.the interface of a second polypeptide) so as to stabilize theheteromultimer, and thereby favor heteromultimer formation overhomomultimer formation, for example. The protuberance may exist in theoriginal interface or may be introduced synthetically (e.g., by alteringnucleic acid encoding the interface). In some embodiments, nucleic acidencoding the interface of the first polypeptide is altered to encode theprotuberance. To achieve this, the nucleic acid encoding at least one“original” amino acid residue in the interface of the first polypeptideis replaced with nucleic acid encoding at least one “import” amino acidresidue which has a larger side chain volume than the original aminoacid residue. It will be appreciated that there can be more than oneoriginal and corresponding import residue. The side chain volumes of thevarious amino residues are shown, for example, in Table 1 ofUS2011/0287009. A mutation to introduce a “protuberance” may be referredto as a “knob mutation.”

In some embodiments, import residues for the formation of a protuberanceare naturally occurring amino acid residues selected from arginine (R),phenylalanine (F), tyrosine (Y) and tryptophan (W). In some embodiments,an import residue is tryptophan or tyrosine. In some embodiment, theoriginal residue for the formation of the protuberance has a small sidechain volume, such as alanine, asparagine, aspartic acid, glycine,serine, threonine or valine.

A “cavity” refers to at least one amino acid side chain which isrecessed from the interface of a second polypeptide and thereforeaccommodates a corresponding protuberance on the adjacent interface of afirst polypeptide. The cavity may exist in the original interface or maybe introduced synthetically (e.g. by altering nucleic acid encoding theinterface). In some embodiments, nucleic acid encoding the interface ofthe second polypeptide is altered to encode the cavity. To achieve this,the nucleic acid encoding at least one “original” amino acid residue inthe interface of the second polypeptide is replaced with DNA encoding atleast one “import” amino acid residue which has a smaller side chainvolume than the original amino acid residue. It will be appreciated thatthere can be more than one original and corresponding import residue. Insome embodiments, import residues for the formation of a cavity arenaturally occurring amino acid residues selected from alanine (A),serine (S), threonine (T) and valine (V). In some embodiments, an importresidue is serine, alanine or threonine. In some embodiments, theoriginal residue for the formation of the cavity has a large side chainvolume, such as tyrosine, arginine, phenylalanine or tryptophan. Amutation to introduce a “cavity” may be referred to as a “holemutation.”

The protuberance is “positionable” in the cavity which means that thespatial location of the protuberance and cavity on the interface of afirst polypeptide and second polypeptide respectively and the sizes ofthe protuberance and cavity are such that the protuberance can belocated in the cavity without significantly perturbing the normalassociation of the first and second polypeptides at the interface. Sinceprotuberances such as Tyr, Phe and Trp do not typically extendperpendicularly from the axis of the interface and have preferredconformations, the alignment of a protuberance with a correspondingcavity may, in some instances, rely on modeling the protuberance/cavitypair based upon a three-dimensional structure such as that obtained byX-ray crystallography or nuclear magnetic resonance (NMR). This can beachieved using widely accepted techniques in the art.

In some embodiments, a knob mutation in an IgG1 constant region is T366W(EU numbering). In some embodiments, a hole mutation in an IgG1 constantregion comprises one or more mutations selected from T366S, L368A andY407V (EU numbering). In some embodiments, a hole mutation in an IgG1constant region comprises T366S, L368A and Y407V (EU numbering).

In some embodiments, a knob mutation in an IgG4 constant region is T366W(EU numbering). In some embodiments, a hole mutation in an IgG4 constantregion comprises one or more mutations selected from T366S, L368A, andY407V (EU numbering). In some embodiments, a hole mutation in an IgG4constant region comprises T366S, L368A, and Y407V (EU numbering).

Multi-specific antibodies may also be made by engineering electrostaticsteering effects for making antibody Fc-heterodimeric molecules (WO2009/089004A 1); cross-linking two or more antibodies or fragments (see,e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229: 81(1985)); using leucine zippers to produce bi-specific antibodies (see,e.g., Kostelny et al., J. Immunol., 148(5): 1547-1553 (1992)); using“diabody” technology for making bispecific antibody fragments (see,e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448(1993)); and using single-chain Fv (sFv) dimers (see, e.g. Gruber etal., J. Immunol., 152:5368 (1994)); and preparing trispecific antibodiesas described, e.g., in Tutt et al. J. Immunol. 147: 60 (1991).

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies,” are also included herein (see,e.g. US 2006/0025576A1).

The antibody or fragment herein also includes a “Dual Acting FAb” or“DAF” comprising an antigen binding site that binds to HER2 as well asanother, different antigen (see, US 2008/0069820, for example).

7. Antibody Variants

In certain embodiments, amino acid sequence variants of the antibodiesprovided herein are contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody. Amino acid sequence variants of an antibody may be prepared byintroducing appropriate modifications into the nucleotide sequenceencoding the antibody, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., antigen-binding.

a) Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the HVRs and FRs. Conservative substitutions areshown in Table 1 under the heading of “preferred substitutions.” Moresubstantial changes are provided in Table 1 under the heading of“exemplary substitutions,” and as further described below in referenceto amino acid side chain classes. Amino acid substitutions may beintroduced into an antibody of interest and the products screened for adesired activity, e.g., retained/improved antigen binding, decreasedimmunogenicity, or improved ADCC or CDC.

TABLE 1 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine;Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

Amino acids may be grouped according to common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antibody and/or will have substantially retainedcertain biological properties of the parent antibody.

An exemplary substitutional variant is an affinity matured antibody,which may be conveniently generated, e.g., using phage display-basedaffinity maturation techniques such as those described herein. Briefly,one or more HVR residues are mutated and the variant antibodiesdisplayed on phage and screened for a particular biological activity(e.g. binding affinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improveantibody affinity. Such alterations may be made in HVR “hotspots,” i.e.,residues encoded by codons that undergo mutation at high frequencyduring the somatic maturation process (see, e.g., Chowdhury, MethodsMol. Biol. 207:179-196 (2008)), and/or SDRs (a-CDRs), with the resultingvariant VH or VL being tested for binding affinity. Affinity maturationby constructing and reselecting from secondary libraries has beendescribed, e.g., in Hoogenboom et al. in Methods in Molecular Biology178:1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., (2001).) Insome embodiments of affinity maturation, diversity is introduced intothe variable genes chosen for maturation by any of a variety of methods(e.g., error-prone PCR, chain shuffling, or oligonucleotide-directedmutagenesis). A secondary library is then created. The library is thenscreened to identify any antibody variants with the desired affinity.Another method to introduce diversity involves HVR-directed approaches,in which several HVR residues (e.g., 4-6 residues at a time) arerandomized. HVR residues involved in antigen binding may be specificallyidentified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3and CDR-L3 in particular are often targeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. Such alterations may be outside of HVR “hotspots” orSDRs. In certain embodiments of the variant VH and VL sequences providedabove, each HVR either is unaltered, or contains no more than one, twoor three amino acid substitutions.

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham and Wells (1989) Science,244:1081-1085. In this method, a residue or group of target residues(e.g., charged residues such as arg, asp, his, lys, and glu) areidentified and replaced by a neutral or negatively charged amino acid(e.g., alanine or polyalanine) to determine whether the interaction ofthe antibody with antigen is affected. Further substitutions may beintroduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, a crystal structure of an antigen-antibody complex is usedto identify contact points between the antibody and antigen. Suchcontact residues and neighboring residues may be targeted or eliminatedas candidates for substitution. Variants may be screened to determinewhether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g. for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

b) Glycosylation Variants

In certain embodiments, an antibody provided herein is altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to an antibody may beconveniently accomplished by altering the amino acid sequence such thatone or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydratestructure that lacks fucose attached (directly or indirectly) to an Fcregion. For example, the amount of fucose in such antibody may be from1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amountof fucose is determined by calculating the average amount of fucosewithin the sugar chain at Asn297, relative to the sum of allglycostructures attached to Asn 297 (e.g. complex, hybrid and highmannose structures) as measured by MALDI-TOF mass spectrometry, asdescribed in WO 2008/077546, for example. Asn297 refers to theasparagine residue located at about position 297 in the Fc region (Eunumbering of Fc region residues); however, Asn297 may also be locatedabout +3 amino acids upstream or downstream of position 297, i.e.,between positions 294 and 300, due to minor sequence variations inantibodies. Such fucosylation variants may have improved ADCC function.See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publicationsrelated to “defucosylated” or “fucose-deficient” antibody variantsinclude: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614;US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki etal. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech.Bioeng. 87: 614 (2004). Examples of cell lines capable of producingdefucosylated antibodies include Lecl3 CHO cells deficient in proteinfucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986);US Pat Appl No US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1,Adams et al., especially at Example 11), and knockout cell lines, suchas alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see,e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. etal., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

Antibodies variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet etal.); U.S. Pat. No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umanaet al.). Antibody variants with at least one galactose residue in theoligosaccharide attached to the Fc region are also provided. Suchantibody variants may have improved CDC function. Such antibody variantsare described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964(Raju, S.); and WO 1999/22764 (Raju, S.).

c) Fe Region Variants

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid modification (e.g. a substitution) atone or more amino acid positions.

In certain embodiments, the invention contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In vitro and/or invivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express Fc(RIII only, whereas monocytes express Fc(RI, Fc(RII andFc(RIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).Non-limiting examples of in vitro assays to assess ADCC activity of amolecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g.Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) andHellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985);U.S. Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med.166:1351-1361 (1987)). Alternatively, non-radioactive assays methods maybe employed (see, for example, ACTI™ non-radioactive cytotoxicity assayfor flow cytometry (CellTechnology, Inc. Mountain View, Calif.; andCytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wis.).Useful effector cells for such assays include peripheral bloodmononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively,or additionally, ADCC activity of the molecule of interest may beassessed in vivo, e.g., in a animal model such as that disclosed inClynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). Clq bindingassays may also be carried out to confirm that the antibody is unable tobind Clq and hence lacks CDC activity. See, e.g., Clq and C3c bindingELISA in WO 2006/029879 and WO 2005/100402. To assess complementactivation, a CDC assay may be performed (see, for example,Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M. S.et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie,Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/halflife determinations can also be performed using methods known in the art(see, e.g., Petkova, S. B. et al., Int'l. Immunol. 18(12):1759-1769(2006)).

In some embodiments, one or more amino acid modifications may beintroduced into the Fc portion of the antibody provided herein in orderto increase IgG binding to the neonatal Fc receptor. In certainembodiments, the antibody comprises the following three mutationsaccording to EU numbering: M252Y, S254T, and T256E (the “YTE mutation”)(U.S. Pat. No. 8,697,650; see also Dall'Acqua et al., Journal ofBiological Chemistry 281(33):23514-23524 (2006). In certain embodiments,the YTE mutation does not affect the ability of the antibody to bind toits cognate antigen. In certain embodiments, the YTE mutation increasesthe antibody's serum half-life compared to the native (i.e., non-YTEmutant) antibody. In some embodiments, the YTE mutation increases theserum half-life of the antibody by 3-fold compared to the native (i.e.,non-YTE mutant) antibody. In some embodiments, the YTE mutationincreases the serum half-life of the antibody by 2-fold compared to thenative (i.e., non-YTE mutant) antibody. In some embodiments, the YTEmutation increases the serum half-life of the antibody by 4-foldcompared to the native (i.e., non-YTE mutant) antibody. In someembodiments, the YTE mutation increases the serum half-life of theantibody by at least 5-fold compared to the native (i.e., non-YTEmutant) antibody. In some embodiments, the YTE mutation increases theserum half-life of the antibody by at least 10-fold compared to thenative (i.e., non-YTE mutant) antibody. See, e.g., U.S. Pat. No.8,697,650; see also Dall'Acqua et al., Journal of Biological Chemistry281(33):23514-23524 (2006).

In certain embodiments, the YTE mutant provides a means to modulateantibody-dependent cell-mediated cytotoxicity (ADCC) activity of theantibody. In certain embodiments, the YTEO mutant provides a means tomodulate ADCC activity of a humanized IgG antibody directed against ahuman antigen. See, e.g., U.S. Pat. No. 8,697,650; see also Dall'Acquaet al., Journal of Biological Chemistry 281(33):23514-23524 (2006).

In certain embodiments, the YTE mutant allows the simultaneousmodulation of serum half-life, tissue distribution, and antibodyactivity (e.g., the ADCC activity of an IgG antibody). See, e.g., U.S.Pat. No. 8,697,650; see also Dall'Acqua et al., Journal of BiologicalChemistry 281(33):23514-23524 (2006).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

In certain embodiments, the proline at position 329 (EU numbering)(P329) of a wild-type human Fc region is substituted with glycine orarginine or an amino acid residue large enough to destroy the prolinesandwich within the Fc/Fc□ receptor interface, that is formed betweenthe P329 of the Fc and tryptophane residues W87 and W110 of FcgRIII(Sondermann et al.: Nature 406, 267-273 (20 Jul. 2000)). In a furtherembodiment, at least one further amino acid substitution in the Fcvariant is S228P, E233P, L234A, L235A, L235E, N297A, N297D, or P331S andstill in another embodiment said at least one further amino acidsubstitution is L234A and L235A of the human IgG1 Fc region or S228P andL235E of the human IgG4 Fc region, all according to EU numbering (U.S.Pat. No. 8,969,526 which is incorporated by reference in its entirety).

In certain embodiments, a polypeptide comprises the Fc variant of awild-type human IgG Fc region wherein the polypeptide has P329 of thehuman IgG Fc region substituted with glycine and wherein the Fc variantcomprises at least two further amino acid substitutions at L234A andL235A of the human IgG1 Fc region or S228P and L235E of the human IgG4Fc region, and wherein the residues are numbered according to the EUnumbering (U.S. Pat. No. 8,969,526 which is incorporated by reference inits entirety). In certain embodiments, the polypeptide comprising theP329G, L234A and L235A (EU numbering) substitutions exhibit a reducedaffinity to the human FcγRIIIA and FcγRIIA, for down-modulation of ADCCto at least 20% of the ADCC induced by the polypeptide comprising thewildtype human IgG Fc region, and/or for down-modulation of ADCP (U.S.Pat. No. 8,969,526 which is incorporated by reference in its entirety).

In a specific embodiment the polypeptide comprising an Fc variant of awildtype human Fc polypeptide comprises a triple mutation: an amino acidsubstitution at position Pro329, a L234A and a L235A mutation accordingto EU numbering (P329/LALA) (U.S. Pat. No. 8,969,526 which isincorporated by reference in its entirety). In specific embodiments, thepolypeptide comprises the following amino acid substitutions: P329G,L234A, and L235A according to EU numbering.

Certain antibody variants with improved or diminished binding to FcRsare described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions which improve ADCC, e.g.,substitutions at positions 298, 333, and/or 334 of the Fc region (EUnumbering of residues).

In some embodiments, alterations are made in the Fc region that resultin altered (i.e., either improved or diminished) Clq binding and/orComplement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat.No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

Antibodies with increased half lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)), are described inUS2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc regionwith one or more substitutions therein which improve binding of the Fcregion to FcRn. Such Fc variants include those with substitutions at oneor more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434,e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).

See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. Nos.5,648,260; 5,624,821; and WO 94/29351 concerning other examples of Fcregion variants.

d) Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., a “THIOMAB™,” in which one or more residuesof an antibody are substituted with cysteine residues. In particularembodiments, the substituted residues occur at sites of the antibodythat are available for conjugation. By substituting those residues withcysteine, reactive thiol groups are thereby positioned at accessiblesites of the antibody and may be used to conjugate the antibody to othermoieties, such as drug moieties or linker-drug moieties, to create animmunoconjugate, as described further herein. In certain embodiments,any one or more of the following residues may be substituted withcysteine: K149 (Kabat numbering) of the light chain; V205 (Kabatnumbering) of the light chain; A 118 (EU numbering) of the heavy chain;A140 (EU numbering) of the heavy chain; L174 (EU numbering) of the heavychain; Y373 (EU numbering) of the heavy chain; and S400 (EU numbering)of the heavy chain Fc region. In specific embodiments, the antibodiesdescribed herein comprise the HC-A140C (EU numbering) cysteinesubstitution. In specific embodiments, the antibodies described hereincomprise the LC-K149C (Kabat numbering) cysteine substitution. Inspecific embodiments, the antibodies described herein comprise theHC-A118C (EU numbering) cysteine substitution. Cysteine engineeredantibodies may be generated as described, e.g., in U.S. Pat. No.7,521,541.

In certain embodiments, the antibody comprises one of the followingheavy chain cysteine substitutions:

Chain EU Mutation Kabat Mutation (HC/LC) Residue Site # Site # HC T 114110 HC A 140 136 HC L 174 170 HC L 179 175 HC T 187 183 HC T 209 205 HCV 262 258 HC G 371 367 HC Y 373 369 HC E 382 378 HC S 424 420 HC N 434430 HC Q 438 434

In certain embodiments, the antibody comprises one of the followinglight chain cysteine substitutions:

Chain EU Mutation Kabat Mutation (HC/LC) Residue Site # Site # LC I 106106 LC R 108 108 LC R 142 142 LC K 149 149 LC V 205 205

A nonlimiting exemplary hu7C2.v2.2.LA light chain (LC) K149C THIOMAB™has the heavy chain and light chain amino acid sequences of SEQ ID NOs:19 and 23, respectively. A nonlimiting exemplary hu7C2.v2.2.LA heavychain (HC) A118C THIOMAB™ has the heavy chain and light chain amino acidsequences of SEQ ID NOs: 24 and 18, respectively.

An exemplary S400C cysteine engineered heavy chain constant region isshown in SEQ ID NO: 28. The S400C cysteine engineered heavy chainconstant region may be fused to the C-terminus of the hu7C2.v2.2.LAheavy chain variable region shown in SEQ ID NO: 11. The resultinghu7C2.v2.2.LA HC S400C heavy chain may be paired with a hu7C2.v2.2.LAkappa light chain, such as the light chain shown in SEQ ID NO: 18.

An exemplary V205C cysteine engineered light chain constant region isshown in SEQ ID NO: 25. The V205C cysteine engineered light chainconstant region may be fused to the C-terminus of the hu7C2.v2.2.LAlight chain variable region shown in SEQ ID NO: 10. The resultinghu7C2.v2.2.LA LC V205C light chain may be paired with a hu7C2.v2.2.LAIgG heavy chain, such as the heavy chain shown in SEQ ID NO: 19.

e) Antibody Derivatives

In certain embodiments, an antibody provided herein may be furthermodified to contain additional nonproteinaceous moieties that are knownin the art and readily available. The moieties suitable forderivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer areattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions,etc.

In another embodiment, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the nonproteinaceous moiety is a carbonnanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605(2005)). The radiation may be of any wavelength, and includes, but isnot limited to, wavelengths that do not harm ordinary cells, but whichheat the nonproteinaceous moiety to a temperature at which cellsproximal to the antibody-nonproteinaceous moiety are killed.

B. Recombinant Methods and Compositions

Antibodies may be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. In one embodiment,isolated nucleic acid encoding an anti-HER2 antibody described herein isprovided. Such nucleic acid may encode an amino acid sequence comprisingthe VL and/or an amino acid sequence comprising the VH of the antibody(e.g., the light and/or heavy chains of the antibody). In a furtherembodiment, one or more vectors (e.g., expression vectors) comprisingsuch nucleic acid are provided. In a further embodiment, a host cellcomprising such nucleic acid is provided. In one such embodiment, a hostcell comprises (e.g., has been transformed with): (1) a vectorcomprising a nucleic acid that encodes an amino acid sequence comprisingthe VL of the antibody and an amino acid sequence comprising the VH ofthe antibody, or (2) a first vector comprising a nucleic acid thatencodes an amino acid sequence comprising the VL of the antibody and asecond vector comprising a nucleic acid that encodes an amino acidsequence comprising the VH of the antibody. In one embodiment, the hostcell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoidcell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of makingan anti-HER2 antibody is provided, wherein the method comprisesculturing a host cell comprising a nucleic acid encoding the antibody,as provided above, under conditions suitable for expression of theantibody, and optionally recovering the antibody from the host cell (orhost cell culture medium).

For recombinant production of an anti-HER2 antibody, nucleic acidencoding an antibody, e.g., as described above, is isolated and insertedinto one or more vectors for further cloning and/or expression in a hostcell. Such nucleic acid may be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J., 2003), pp. 245-254, describing expression of antibody fragments inE. coli.) After expression, the antibody may be isolated from thebacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li etal., Nat. Biotech. 24:210-215 (2006).

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977));baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkeykidney cells (CV 1); African green monkey kidney cells (VERO-76); humancervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo ratliver cells (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., inMather et al., Annals N. Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells;and FS4 cells. Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al.,Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines suchas Y0, NS0 and Sp2/0. For a review of certain mammalian host cell linessuitable for antibody production, see, e.g., Yazaki and Wu, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J.), pp. 255-268 (2003).

C. Assays

Anti-HER2 antibodies provided herein may be identified, screened for, orcharacterized for their physical/chemical properties and/or biologicalactivities by various assays known in the art.

In one aspect, an antibody of the invention is tested for its antigenbinding activity, e.g., by known methods such as ELISA, BIACore®, FACS,or Western blot.

In another aspect, competition assays may be used to identify anantibody that competes with any of the antibodies described herein forbinding to HER2. In certain embodiments, such a competing antibody bindsto the same epitope (e.g., a linear or a conformational epitope) that isbound by an antibody described herein. Detailed exemplary methods formapping an epitope to which an antibody binds are provided in Morris(1996) “Epitope Mapping Protocols,” in Methods in Molecular Biology vol.66 (Humana Press, Totowa, N.J.).

In an exemplary competition assay, immobilized HER2 is incubated in asolution comprising a first labeled antibody that binds to HER2 (e.g.,any of the antibodies described herein) and a second unlabeled antibodythat is being tested for its ability to compete with the first antibodyfor binding to HER2. The second antibody may be present in a hybridomasupernatant. As a control, immobilized HER2 is incubated in a solutioncomprising the first labeled antibody but not the second unlabeledantibody. After incubation under conditions permissive for binding ofthe first antibody to HER2, excess unbound antibody is removed, and theamount of label associated with immobilized HER2 is measured. If theamount of label associated with immobilized HER2 is substantiallyreduced in the test sample relative to the control sample, then thatindicates that the second antibody is competing with the first antibodyfor binding to HER2. See Harlow and Lane (1988) Antibodies: A LaboratoryManual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

D. Immunoconjugates

The invention also provides immunoconjugates comprising any anti-HER2antibody provided herein conjugated to one or more cytotoxic agents,such as chemotherapeutic agents or drugs, growth inhibitory agents,toxins (e.g., protein toxins, enzymatically active toxins of bacterial,fungal, plant, or animal origin, or fragments thereof), or radioactiveisotopes (i.e., a radioconjugate).

Immunoconjugates allow for the targeted delivery of a drug moiety to atumor, and, in some embodiments intracellular accumulation therein,where systemic administration of unconjugated drugs may result inunacceptable levels of toxicity to normal cells (Polakis P. (2005)Current Opinion in Pharmacology 5:382-387).

Antibody-drug conjugates (ADC) are targeted chemotherapeutic moleculeswhich combine properties of both antibodies and cytotoxic drugs bytargeting potent cytotoxic drugs to antigen-expressing tumor cells(Teicher, B. A. (2009) Current Cancer Drug Targets 9:982-1004), therebyenhancing the therapeutic index by maximizing efficacy and minimizingoff-target toxicity (Carter, P. J. and Senter P. D. (2008) The CancerJour. 14(3):154-169; Chari, R. V. (2008) Ace. Chem. Res. 41:98-107.

The ADC compounds of the invention include those with anticanceractivity. In some embodiments, the ADC compounds include an antibodyconjugated, i.e. covalently attached, to the drug moiety. In someembodiments, the antibody is covalently attached to the drug moietythrough a linker. The antibody-drug conjugates (ADC) of the inventionselectively deliver an effective dose of a drug to tumor tissue wherebygreater selectivity, i.e. a lower efficacious dose, may be achievedwhile increasing the therapeutic index (“therapeutic window”).

The drug moiety (D) of the antibody-drug conjugates (ADC) may includeany compound, moiety or group that has a cytotoxic or cytostatic effect.Drug moieties may impart their cytotoxic and cytostatic effects bymechanisms including but not limited to tubulin binding, DNA binding orintercalation, and inhibition of RNA polymerase, protein synthesis,and/or topoisomerase.

Exemplary drug moieties include, but are not limited to,pyrrolobenzodiazepine (PBD), and stereoisomers, isosteres, analogs, andderivatives thereof that have cytotoxic activity. Nonlimiting examplesof such immunoconjugates are discussed in further detail below.

1. Exemplary Antibody-Drug Conjugates

An exemplary embodiment of an antibody-drug conjugate (ADC) compoundcomprises an antibody (Ab) which targets a tumor cell, a drug moiety(D), and a linker moiety (L) that attaches Ab to D. In some embodiments,the antibody is attached to the linker moiety (L) through one or moreamino acid residues, such as lysine and/or cysteine.

An exemplary ADC has Formula I:

Ab-(L-D)_(p)  I

where p is 1 to about 20. In some embodiments, the number of drugmoieties that can be conjugated to an antibody is limited by the numberof free cysteine residues. In some embodiments, free cysteine residuesare introduced into the antibody amino acid sequence by the methodsdescribed herein. Exemplary ADC of Formula I include, but are notlimited to, antibodies that have 1, 2, 3, or 4 engineered cysteine aminoacids (Lyon, R. et al (2012) Methods in Enzym. 502:123-138). In someembodiments, one or more free cysteine residues are already present inan antibody, without the use of engineering, in which case the existingfree cysteine residues may be used to conjugate the antibody to a drug.In some embodiments, an antibody is exposed to reducing conditions priorto conjugation of the antibody in order to generate one or more freecysteine residues.

a) Exemplary Linkers

A “Linker” (L) is a bifunctional or multifunctional moiety that can beused to link one or more drug moieties (D) to an antibody (Ab) to forman antibody-drug conjugate (ADC) of Formula I. In some embodiments,antibody-drug conjugates (ADC) can be prepared using a Linker havingreactive functionalities for covalently attaching to the drug and to theantibody. For example, in some embodiments, a cysteine thiol of anantibody (Ab) can form a bond with a reactive functional group of alinker or a drug-linker intermediate to make an ADC.

In one aspect, a linker has a functionality that is capable of reactingwith a free cysteine present on an antibody to form a covalent bond.Nonlimiting exemplary such reactive functionalities include maleimide,haloacetamides, α-haloacetyl, activated esters such as succinimideesters, 4-nitrophenyl esters, pentafluorophenyl esters,tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonylchlorides, isocyanates, and isothiocyanates. See, e.g., the conjugationmethod at page 766 of Klussman, et al (2004), Bioconjugate Chemistry15(4):765-773, and the Examples herein.

In some embodiments, a linker has a functionality that is capable ofreacting with an electrophilic group present on an antibody. Exemplarysuch electrophilic groups include, but are not limited to, aldehyde andketone carbonyl groups. In some embodiments, a heteroatom of thereactive functionality of the linker can react with an electrophilicgroup on an antibody and form a covalent bond to an antibody unit.Nonlimiting exemplary such reactive functionalities include, but are notlimited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone,hydrazine carboxylate, and arylhydrazide.

A linker may comprise one or more linker components. Exemplary linkercomponents include 6-maleimidocaproyl (“MC”), maleimidopropanoyl (“MP”),valine-citrulline (“val-cit” or “vc”), alanine-phenylalanine(“ala-phe”), p-aminobenzyloxycarbonyl (a “PAB”), N-Succinimidyl4-(2-pyridylthio) pentanoate (“SPP”), and 4-(N-maleimidomethyl)cyclohexane-1 carboxylate (“MCC”). Various linker components are knownin the art, some of which are described below.

A linker may be a “cleavable linker,” facilitating release of a drug.Nonlimiting exemplary cleavable linkers include acid-labile linkers(e.g., comprising hydrazone), protease-sensitive (e.g.,peptidase-sensitive) linkers, photolabile linkers, ordisulfide-containing linkers (Chari et al., Cancer Research 52:127-131(1992); U.S. Pat. No. 5,208,020).

In certain embodiments, a linker has the following Formula II:

-A_(a)-W_(w)—Y_(y)—  II

wherein A is a “stretcher unit”, and a is an integer from 0 to 1; W isan “amino acid unit”, and w is an integer from 0 to 12; Y is a “spacerunit”, and y is 0, 1, or 2; and Ab, D, and p are defined as above forFormula I. Exemplary embodiments of such linkers are described in U.S.Pat. No. 7,498,298, which is expressly incorporated herein by reference.

In some embodiments, a linker component comprises a “stretcher unit”that links an antibody to another linker component or to a drug moiety.Nonlimiting exemplary stretcher units are shown below (wherein the wavyline indicates sites of covalent attachment to an antibody, drug, oradditional linker components):

In some embodiments, a linker component comprises an “amino acid unit”.In some such embodiments, the amino acid unit allows for cleavage of thelinker by a protease, thereby facilitating release of the drug from theimmunoconjugate upon exposure to intracellular proteases, such aslysosomal enzymes (Doronina et al. (2003) Nat. Biotechnol. 21:778-784).Exemplary amino acid units include, but are not limited to, dipeptides,tripeptides, tetrapeptides, and pentapeptides. Exemplary dipeptidesinclude, but are not limited to, valine-citrulline (vc or val-cit),alanine-phenylalanine (af or ala-phe); phenylalanine-lysine (fk orphe-lys); phenylalanine-homolysine (phe-homolys); andN-methyl-valine-citrulline (Me-val-cit). Exemplary tripeptides include,but are not limited to, glycine-valine-citrulline (gly-val-cit) andglycine-glycine-glycine (gly-gly-gly). An amino acid unit may compriseamino acid residues that occur naturally and/or minor amino acids and/ornon-naturally occurring amino acid analogs, such as citrulline. Aminoacid units can be designed and optimized for enzymatic cleavage by aparticular enzyme, for example, a tumor-associated protease, cathepsinB, C and D, or a plasmin protease.

In some embodiments, a linker component comprises a “spacer” unit thatlinks the antibody to a drug moiety, either directly or through astretcher unit and/or an amino acid unit. A spacer unit may be“self-immolative” or a “non-self-immolative.” A “non-self-immolative”spacer unit is one in which part or all of the spacer unit remains boundto the drug moiety upon cleavage of the ADC. Examples ofnon-self-immolative spacer units include, but are not limited to, aglycine spacer unit and a glycine-glycine spacer unit. In someembodiments, enzymatic cleavage of an ADC containing a glycine-glycinespacer unit by a tumor-cell associated protease results in release of aglycine-glycine-drug moiety from the remainder of the ADC. In some suchembodiments, the glycine-glycine-drug moiety is subjected to ahydrolysis step in the tumor cell, thus cleaving the glycine-glycinespacer unit from the drug moiety.

A “self-immolative” spacer unit allows for release of the drug moiety.In certain embodiments, a spacer unit of a linker comprises ap-aminobenzyl unit. In some such embodiments, a p-aminobenzyl alcohol isattached to an amino acid unit via an amide bond, and a carbamate,methylcarbamate, or carbonate is made between the benzyl alcohol and thedrug (Hamann et al. (2005) Expert Opin. Ther. Patents (2005)15:1087-1103). In some embodiments, the spacer unit isp-aminobenzyloxycarbonyl (PAB). In some embodiments, an ADC comprising aself-immolative linker has the structure:

wherein Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro, or -cyno;m is an integer ranging from 0 to 4; and p ranges from 1 to about 20. Insome embodiments, p ranges from 1 to 10, 1 to 7, 1 to 5, or 1 to 4.

Other examples of self-immolative spacers include, but are not limitedto, aromatic compounds that are electronically similar to the PAB group,such as 2-aminoimidazol-5-methanol derivatives (U.S. Pat. No. 7,375,078;Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237) and ortho- orpara-aminobenzylacetals. In some embodiments, spacers can be used thatundergo cyclization upon amide bond hydrolysis, such as substituted andunsubstituted 4-aminobutyric acid amides (Rodrigues et al (1995)Chemistry Biology 2:223), appropriately substituted bicyclo[2.2.1] andbicyclo[2.2.2] ring systems (Storm et al (1972) J. Amer. Chem. Soc.94:5815) and 2-aminophenylpropionic acid amides (Amsberry, et al (1990)J. Org. Chem. 55:5867). Linkage of a drug to the α-carbon of a glycineresidue is another example of a self-immolative spacer that may beuseful in ADC (Kingsbury et al (1984) J. Med. Chem. 27:1447).

In some embodiments, linker L may be a dendritic type linker forcovalent attachment of more than one drug moiety to an antibody througha branching, multifunctional linker moiety (Sun et al (2002) Bioorganic& Medicinal Chemistry Letters 12:2213-2215; Sun et al (2003) Bioorganic& Medicinal Chemistry 11:1761-1768). Dendritic linkers can increase themolar ratio of drug to antibody, i.e. loading, which is related to thepotency of the ADC. Thus, where an antibody bears only one reactivecysteine thiol group, a multitude of drug moieties may be attachedthrough a dendritic linker.

Nonlimiting exemplary linkers are shown below in the context of an ADCof Formula I:

Further nonlimiting exemplary ADCs include the structures:

where X is:

Y is:

each R is independently H or C₁-C₆ alkyl; and n is 1 to 12.

Typically, peptide-type linkers can be prepared by forming a peptidebond between two or more amino acids and/or peptide fragments. Suchpeptide bonds can be prepared, for example, according to a liquid phasesynthesis method (e.g., E. Schröder and K. Lübke (1965) “The Peptides”,volume 1, pp 76-136, Academic Press).

In some embodiments, a linker is substituted with groups that modulatesolubility and/or reactivity. As a nonlimiting example, a chargedsubstituent such as sulfonate (—SO₃ ⁻) or ammonium may increase watersolubility of the linker reagent and facilitate the coupling reaction ofthe linker reagent with the antibody and/or the drug moiety, orfacilitate the coupling reaction of Ab-L (antibody-linker intermediate)with D, or D-L (drug-linker intermediate) with Ab, depending on thesynthetic route employed to prepare the ADC. In some embodiments, aportion of the linker is coupled to the antibody and a portion of thelinker is coupled to the drug, and then the Ab-(linker portion)^(a) iscoupled to drug-(linker portion)^(b) to form the ADC of Formula I. Insome such embodiments, the antibody comprises more than one (linkerportion)^(a) substituents, such that more than one drug is coupled tothe antibody in the ADC of Formula I.

The compounds of the invention expressly contemplate, but are notlimited to, ADC prepared with the following linker reagents:bis-maleimido-trioxyethylene glycol (BMPEO),N-(β-maleimidopropyloxy)-N-hydroxy succinimide ester (BMPS),N-(ε-maleimidocaproyloxy) succinimide ester (EMCS),N-[γ-maleimidobutyryloxy]succinimide ester (GMBS),1,6-hexane-bis-vinylsulfone (HBVS), succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate) (LC-SMCC),m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),4-(4-N-Maleimidophenyl)butyric acid hydrazide (MPBH), succinimidyl3-(bromoacetamido)propionate (SBAP), succinimidyl iodoacetate (SIA),succinimidyl (4-iodoacetyl)aminobenzoate (SIAB),N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP), succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), succinimidyl4-(p-maleimidophenyl)butyrate (SMPB), succinimidyl6-[(beta-maleimidopropionamido)hexanoate](SMPH), iminothiolane (IT),sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC,and sulfo-SMPB, and succinimidyl-(4-vinylsulfone)benzoate (SVSB), andincluding bis-maleimide reagents: dithiobismaleimidoethane (DTME),1,4-Bismaleimidobutane (BMB), 1,4 Bismaleimidyl-2,3-dihydroxybutane(BMDB), bismaleimidohexane (BMH), bismaleimidoethane (BMOE), BM(PEG)₂(shown below), and BM(PEG)₃ (shown below); bifunctional derivatives ofimidoesters (such as dimethyl adipimidate HCl), active esters (such asdisuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azidocompounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazoniumderivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),diisocyanates (such as toluene 2,6-diisocyanate), and bis-activefluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). In someembodiments, bis-maleimide reagents allow the attachment of the thiolgroup of a cysteine in the antibody to a thiol-containing drug moiety,linker, or linker-drug intermediate. Other functional groups that arereactive with thiol groups include, but are not limited to,iodoacetamide, bromoacetamide, vinyl pyridine, disulfide, pyridyldisulfide, isocyanate, and isothiocyanate.

Certain useful linker reagents can be obtained from various commercialsources, such as Pierce Biotechnology, Inc. (Rockford, Ill.), MolecularBiosciences Inc. (Boulder, Colo.), or synthesized in accordance withprocedures described in the art; for example, in Toki et al (2002) J.Org. Chem. 67:1866-1872; Dubowchik, et al. (1997) Tetrahedron Letters,38:5257-60; Walker, M. A. (1995) J. Org. Chem. 60:5352-5355; Frisch etal (1996) Bioconjugate Chem. 7:180-186; U.S. Pat. No. 6,214,345; WO02/088172; US 2003130189; US2003096743; WO 03/026577; WO 03/043583; andWO 04/032828.

Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See, e.g., WO94/11026.

b) Exemplary Drug Moieties

In some embodiments, an ADC comprises a pyrrolobenzodiazepine (PBD). Insome embodiments, PBD dimers recognize and bind to specific DNAsequences. The natural product anthramycin, a PBD, was first reported in1965 (Leimgruber, et al., (1965) J. Am. Chem. Soc., 87:5793-5795;Leimgruber, et al., (1965) J. Am. Chem. Soc., 87:5791-5793). Since then,a number of PBDs, both naturally-occurring and analogues, have beenreported (Thurston, et al., (1994) Chem. Rev. 1994, 433-465 includingdimers of the tricyclic PBD scaffold (U.S. Pat. Nos. 6,884,799;7,049,311; 7,067,511; 7,265,105; 7,511,032; 7,528,126; 7,557,099).Without intending to be bound by any particular theory, it is believedthat the dimer structure imparts the appropriate three-dimensional shapefor isohelicity with the minor groove of B-form DNA, leading to a snugfit at the binding site (Kohn, In Antibiotics III. Springer-Verlag, NewYork, pp. 3-11 (1975); Hurley and Needham-VanDevanter, (1986) Acc. Chem.Res., 19:230-237). Dimeric PBD compounds bearing C2 aryl substituentshave been shown to be useful as cytotoxic agents (Hartley et al (2010)Cancer Res. 70(17):6849-6858; Antonow (2010) J. Med. Chem.53(7):2927-2941; Howard et al (2009) Bioorganic and Med. Chem. Letters19(22):6463-6466).

In some embodiments, PBD compounds can be employed as prodrugs byprotecting them at the N10 position with a nitrogen protecting groupwhich is removable in vivo (WO 00/12507; WO 2005/023814).

PBD dimers have been conjugated to antibodies and the resulting ADCshown to have anti-cancer properties (US 2010/0203007). Nonlimitingexemplary linkage sites on the PBD dimer include the five-memberedpyrrolo ring, the tether between the PBD units, and the N10-C11 iminegroup (WO 2009/016516; US 2009/304710; US 2010/047257; US 2009/036431;US 2011/0256157; WO 2011/130598).

Nonlimiting exemplary PBD dimer components of ADCs are center-linkedpyrrolobenzodiazepines of Formula A:

wherein:

R² is

where R^(36a) and R^(36b) are independently selected from H, F, C₁₋₄saturated alkyl, C₂₋₃ alkenyl, which alkyl and alkenyl groups areoptionally substituted by a group selected from C₁₋₄ alkyl amido andC₁₋₄ alkyl ester; or, when one of R^(36a) and R^(36b) is H, the other isselected from nitrile and a C₁₋₄ alkyl ester;

R⁶ and R⁹ are independently selected from H, R, OH, OR, SH, SR, NH₂,NHR, NRR′, NO₂, Me₃Sn and halo;

R⁷ is independently selected from H, R, OH, OR, SH, SR, NH₂, NHR, NRR′,NO₂, Me₃Sn and halo;

Y has the formula:

G comprises a reactive group for connecting to an antibody or G is alinker connected to an antibody;

n is an integer selected in the range of 0 to 48;

R^(A4) is a C₁₋₆ alkylene group;

either

-   -   (a) R¹⁰ is H, and R¹¹ is OH, OR^(A), where R^(A) is C₁₋₄ alkyl;        or    -   (b) R¹⁰ and R¹¹ form a nitrogen-carbon double bond between the        nitrogen and carbon atoms to which they are bound; or    -   (c) R⁰ is H and R¹¹ is OSO_(z)M, where z is 2 or 3 and M is a        monovalent pharmaceutically acceptable cation;

R and R′ are each independently selected from optionally substitutedC₁₋₁₂ alkyl, C₃₋₂₀ heterocyclyl and C₅₋₂₀ aryl groups, and optionally inrelation to the group NRR′, R and R′ together with the nitrogen atom towhich they are attached form an optionally substituted 4-, 5-, 6- or7-membered heterocyclic ring;

wherein R¹⁶, R¹⁷, R¹⁹, R²⁰, R²¹ and R²² are as defined for R⁶, R⁷, R⁹,R¹⁰, R¹¹ and R² respectively;

wherein Z is CH or N;

wherein T and T″ are independently selected from a single bond or a C₁₋₉alkylene, which chain may be interrupted by one or more heteroatoms e.g.O, S, N(H), NMe, provided that the number of atoms in the shortest chainof atoms between X and X′ is 3 to 12 atoms; and

X and X′ are independently selected from O, S and N(H).

In some embodiments, R⁹ and R¹⁹ are H.

In some embodiments, R⁶ and R¹⁶ are H.

In some embodiments, R⁷ are R¹⁷ are both OR^(7A), where R^(7A) isoptionally substituted C₁₋₄ alkyl. In some embodiments, R^(7A) is Me. Insome embodiments, R^(7A) is is Ch₂Ph, where Ph is a phenyl group.

In some embodiments, X is O.

In some embodiments, T is selected from a single bond, C₁, and a C₂alkylene group. In some embodiments, T is a C₁ alkylene group.

In some embodiments, R^(36a) and R^(36b) are both H. In someembodiments, R^(36a) and R^(36b) are both methyl. In some embodiments,one of R^(36a) and R^(36b) is H, and the other is selected from C₁₋₄saturated alkyl, C₂₋₃ alkenyl, which alkyl and alkenyl groups areoptionally substituted. In some embodiments, the group of R^(36a) andR^(36b) which is not H is selected from methyl and ethyl.

In some embodiments, R¹⁰ is H and R¹¹ is OH. In some embodiments, R¹⁰and R¹¹ form a nitrogen-carbon double bond between the nitrogen andcarbon atoms to which they are bound.

In any of the embodiments described herein, R¹⁶, R¹⁷, R¹⁹, R²⁰, R²¹,R²², X′ and T′ may be the same as R⁶, R⁷, R⁹, R¹⁰, R¹¹, R², X and Trespectively.

In some embodiments, G comprises a group selected from (i) maleimidegroups (ii) activated disulfides, (iii) active esters such as NHS(N-hydroxysuccinimide) esters, HOBt (N-hydroxybenzotriazole) esters,haloformates, and acid halides; (iv) alkyl and benzyl halides such ashaloacetamides; and (v) aldehydes, ketones, carboxyl comprises amaleimide group, an activated disulfide, or an electrophilic functionalgroup. In some embodiments, G is a linker connected to an antibody,wherein the linker comprises a moiety derived from an electrophilicfunctional group selected from (i) maleimide groups (ii) activateddisulfides, (iii) active esters such as NHS (N-hydroxysuccinimide)esters, HOBt (N-hydroxybenzotriazole) esters, haloformates, and acidhalides; (iv) alkyl and benzyl halides such as haloacetamides; and (v)aldehydes, ketones, carboxyl. In some embodiments, G is:

wherein the wavy line indicates attachment to the remainder of (A1). Insome embodiments, G is:

wherein the wavy line indicates attachment to the remainder of (A1) andAb is an antibody.

In some embodiments, an ADC comprises a center-linked PBD comprising thestructure:

wherein Y is defined as above.

A non-limiting exemplary ADC comprising a center-linked PBD dimer may bemade by conjugating a center-linked PBD drug linker intermediate (shownbelow) to an antibody:

to produce a center-linked PBD antibody-drug conjugate:

Another center-linked PBD drug linker intermediate is:

which is conjugated to an antibody to form an ADC:

PBD dimers and ADCs comprising PBD dimers may be prepared according tomethods known in the art. See, e.g., WO 2009/016516; US 2009/304710; US2010/047257; US 2009/036431; US 2011/0256157; WO 2011/130598; WO2013/055987; WO 2014/159981; WO 2014/140862.

c) Drug Loading

Drug loading is represented by p, the average number of drug moietiesper antibody in a molecule of Formula I. Drug loading may range from 1to 20 drug moieties (D) per antibody. ADCs of Formula I includecollections of antibodies conjugated with a range of drug moieties, from1 to 20. The average number of drug moieties per antibody inpreparations of ADC from conjugation reactions may be characterized byconventional means such as mass spectroscopy, ELISA assay, and HPLC. Thequantitative distribution of ADC in terms of p may also be determined.In some instances, separation, purification, and characterization ofhomogeneous ADC where p is a certain value from ADC with other drugloadings may be achieved by means such as reverse phase HPLC orelectrophoresis.

For some antibody-drug conjugates, p may be limited by the number ofattachment sites on the antibody. For example, where the attachment is acysteine thiol, as in certain exemplary embodiments above, an antibodymay have only one or several cysteine thiol groups, or may have only oneor several sufficiently reactive thiol groups through which a linker maybe attached. In certain embodiments, higher drug loading, e.g. p>5, maycause aggregation, insolubility, toxicity, or loss of cellularpermeability of certain antibody-drug conjugates. In certainembodiments, the average drug loading for an ADC ranges from 1 to about8; from about 2 to about 6; or from about 3 to about 5. Indeed, it hasbeen shown that for certain ADCs, the optimal ratio of drug moieties perantibody may be less than 8, and may be about 2 to about 5 (U.S. Pat.No. 7,498,298).

In certain embodiments, fewer than the theoretical maximum of drugmoieties are conjugated to an antibody during a conjugation reaction. Anantibody may contain, for example, lysine residues that do not reactwith the drug-linker intermediate or linker reagent, as discussed below.Generally, antibodies do not contain many free and reactive cysteinethiol groups which may be linked to a drug moiety; indeed most cysteinethiol residues in antibodies exist as disulfide bridges. In certainembodiments, an antibody may be reduced with a reducing agent such asdithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), under partialor total reducing conditions, to generate reactive cysteine thiolgroups. In certain embodiments, an antibody is subjected to denaturingconditions to reveal reactive nucleophilic groups such as lysine orcysteine.

The loading (drug/antibody ratio) of an ADC may be controlled indifferent ways, and for example, by: (i) limiting the molar excess ofdrug-linker intermediate or linker reagent relative to antibody, (ii)limiting the conjugation reaction time or temperature, and (iii) partialor limiting reductive conditions for cysteine thiol modification.

It is to be understood that where more than one nucleophilic groupreacts with a drug-linker intermediate or linker reagent, then theresulting product is a mixture of ADC compounds with a distribution ofone or more drug moieties attached to an antibody. The average number ofdrugs per antibody may be calculated from the mixture by a dual ELISAantibody assay, which is specific for antibody and specific for thedrug. Individual ADC molecules may be identified in the mixture by massspectroscopy and separated by HPLC, e.g. hydrophobic interactionchromatography (see, e.g., McDonagh et al (2006) Prot. Engr. Design &Selection 19(7):299-307; Hamblett et al (2004) Clin. Cancer Res.10:7063-7070; Hamblett, K. J., et al. “Effect of drug loading on thepharmacology, pharmacokinetics, and toxicity of an anti-CD30antibody-drug conjugate,” Abstract No. 624, American Association forCancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings ofthe AACR, Volume 45, March 2004; Alley, S. C., et al. “Controlling thelocation of drug attachment in antibody-drug conjugates,” Abstract No.627, American Association for Cancer Research, 2004 Annual Meeting, Mar.27-31, 2004, Proceedings of the AACR, Volume 45, March 2004). In certainembodiments, a homogeneous ADC with a single loading value may beisolated from the conjugation mixture by electrophoresis orchromatography.

d) Certain Methods of Preparing Immunoconjugates

An ADC of Formula I may be prepared by several routes employing organicchemistry reactions, conditions, and reagents known to those skilled inthe art, including: (1) reaction of a nucleophilic group of an antibodywith a bivalent linker reagent to form Ab-L via a covalent bond,followed by reaction with a drug moiety D; and (2) reaction of anucleophilic group of a drug moiety with a bivalent linker reagent, toform D-L, via a covalent bond, followed by reaction with a nucleophilicgroup of an antibody. Exemplary methods for preparing an ADC of FormulaI via the latter route are described in U.S. Pat. No. 7,498,298, whichis expressly incorporated herein by reference.

Nucleophilic groups on antibodies include, but are not limited to:(i)N-terminal amine groups, (ii) side chain amine groups, e.g. lysine,(iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl oramino groups where the antibody is glycosylated. Amine, thiol, andhydroxyl groups are nucleophilic and capable of reacting to formcovalent bonds with electrophilic groups on linker moieties and linkerreagents including: (i) active esters such as NHS esters, HOBt esters,haloformates, and acid halides; (ii) alkyl and benzyl halides such ashaloacetamides; and (iii) aldehydes, ketones, carboxyl, and maleimidegroups. Certain antibodies have reducible interchain disulfides, i.e.cysteine bridges. Antibodies may be made reactive for conjugation withlinker reagents by treatment with a reducing agent such as DTT(dithiothreitol) or tricarbonylethylphosphine (TCEP), such that theantibody is fully or partially reduced. Each cysteine bridge will thusform, theoretically, two reactive thiol nucleophiles. Additionalnucleophilic groups can be introduced into antibodies throughmodification of lysine residues, e.g., by reacting lysine residues with2-iminothiolane (Traut's reagent), resulting in conversion of an amineinto a thiol. Reactive thiol groups may also be introduced into anantibody by introducing one, two, three, four, or more cysteine residues(e.g., by preparing variant antibodies comprising one or more non-nativecysteine amino acid residues).

Antibody-drug conjugates of the invention may also be produced byreaction between an electrophilic group on an antibody, such as analdehyde or ketone carbonyl group, with a nucleophilic group on a linkerreagent or drug. Useful nucleophilic groups on a linker reagent include,but are not limited to, hydrazide, oxime, amino, hydrazine,thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. In oneembodiment, an antibody is modified to introduce electrophilic moietiesthat are capable of reacting with nucleophilic substituents on thelinker reagent or drug. In another embodiment, the sugars ofglycosylated antibodies may be oxidized, e.g. with periodate oxidizingreagents, to form aldehyde or ketone groups which may react with theamine group of linker reagents or drug moieties. The resulting imineSchiff base groups may form a stable linkage, or may be reduced, e.g. byborohydride reagents to form stable amine linkages. In one embodiment,reaction of the carbohydrate portion of a glycosylated antibody witheither galactose oxidase or sodium meta-periodate may yield carbonyl(aldehyde and ketone) groups in the antibody that can react withappropriate groups on the drug (Hermanson, Bioconjugate Techniques). Inanother embodiment, antibodies containing N-terminal serine or threonineresidues can react with sodium meta-periodate, resulting in productionof an aldehyde in place of the first amino acid (Geoghegan & Stroh,(1992) Bioconjugate Chem. 3:138-146; U.S. Pat. No. 5,362,852). Such analdehyde can be reacted with a drug moiety or linker nucleophile.

Exemplary nucleophilic groups on a drug moiety include, but are notlimited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine,thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groupscapable of reacting to form covalent bonds with electrophilic groups onlinker moieties and linker reagents including: (i) active esters such asNHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl andbenzyl halides such as haloacetamides; (iii) aldehydes, ketones,carboxyl, and maleimide groups.

Nonlimiting exemplary cross-linker reagents that may be used to prepareADC are described herein in the section titled “Exemplary Linkers.”Methods of using such cross-linker reagents to link two moieties,including a proteinaceous moiety and a chemical moiety, are known in theart. In some embodiments, a fusion protein comprising an antibody and acytotoxic agent may be made, e.g., by recombinant techniques or peptidesynthesis. A recombinant DNA molecule may comprise regions encoding theantibody and cytotoxic portions of the conjugate either adjacent to oneanother or separated by a region encoding a linker peptide which doesnot destroy the desired properties of the conjugate.

In yet another embodiment, an antibody may be conjugated to a “receptor”(such as streptavidin) for utilization in tumor pre-targeting whereinthe antibody-receptor conjugate is administered to the patient, followedby removal of unbound conjugate from the circulation using a clearingagent and then administration of a “ligand” (e.g., avidin) which isconjugated to a cytotoxic agent (e.g., a drug or radionucleotide).

E. Trastuzumab-MCC-DM1 and Pertuzumab

Trastuzumab-MCC-DM1 (T-DM1)

The present invention includes therapeutic treatments withtrastuzumab-MCC-DM1 (T-DM1, also referred to as trastuzumab emtansine),an antibody-drug conjugate (CAS Reg. No. 139504-50-0), which has thestructure:

where Tr is trastuzumab linked through linker moiety MCC to themaytansinoid drug moiety DM1 (U.S. Pat. Nos. 5,208,020; 6,441,163). Thedrug to antibody ratio or drug loading is represented by p in the abovestructure of trastuzumab-MCC-DM1, and ranges in integer values from 1 toabout 8. Trastuzumab-MCC-DM1 includes all mixtures of variously loadedand attached antibody-drug conjugates where 1, 2, 3, 4, 5, 6, 7, and 8drug moieties are covalently attached to the antibody trastuzumab (U.S.Pat. Nos. 7,097,840; 8,337,856; US 2005/0276812; US 2005/0166993).

Trastuzumab can be produced by a mammalian cell (Chinese Hamster Ovary,CHO) suspension culture. The HER2 (or c-erbB2) proto-oncogene encodes atransmembrane receptor protein of 185 kDa, which is structurally relatedto the epidermal growth factor receptor. Trastuzumab is an antibody thathas antigen binding residues of, or derived from, the murine 4D5antibody (ATCC CRL 10463, deposited with American Type CultureCollection, 12301 Parklawn Drive, Rockville, Md. 20852 under theBudapest Treaty on May 24, 1990). Exemplary humanized 4D5 antibodiesinclude huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5,huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 (trastuzumab, HERCEPTIN) as inU.S. Pat. No. 5,821,337. In some embodiments, the antibody portion ofT-DM 1 comprises the light and heavy chain amino acid sequences shown inSEQ ID NO: 30 and SEQ ID NO. 29, respectively.

Trastuzumab-MCC-DM1 may be prepared according to Example 1 of U.S.Application Publication No. 20110165155, for example.

As a general proposition, the initial pharmaceutically effective amountof trastuzumab-MCC-DM1 administered per dose will be in the range ofabout 0.3 to 15 mg/kg/day of patient body weight.

A commercial T-DM1 formulation (KADCYLA®, ado-trastuzumab emtansine) isa sterile, white to off-white preservative free lyophilized powder insingle-use vials. Each vial contains 100 mg or 160 mg ado-trastuzumabemtansine. Following reconstitution, each single-use vial containsado-trastuzumab emtansine (20 mg/mL), polysorbate 20 [0.02% (w/v)],sodium succinate (10 mM), and sucrose [6% (w/v)] with a pH of 5.0 anddensity of 1.026 g/mL. The resulting solution containing 20 mg/mLado-trastuzumab emtansine is administered by intravenous infusionfollowing dilution. In some embodiments, ado-trastuzumab emtansine isadministered at a dose of 3.6 mg/kg every three weeks. In someembodiments, ado-trastuzumab emtansine is administered at a dose of 2.4mg/kg every week.

Pertuzumab Compositions

The pertuzumab composition comprises a mixture of a main speciespertuzumab antibody, as hereinabove defined, and one or more variantsthereof. The preferred embodiment herein of a pertuzumab main speciesantibody is one comprising a light chain amino acid sequence of SEQ IDNO: 32, and a heavy chain amino acid sequence of SEQ ID NO: 31(including deamidated and/or oxidized variants of those sequences). Insome embodiments, the composition comprises a mixture of the mainspecies pertuzumab antibody and an amino acid sequence variant thereofcomprising an amino-terminal leader extension, e.g., comprising a lightchain amino acid sequence of SEQ ID NO: 34, and a heavy chain amino acidsequence of SEQ ID NO: 33. Preferably, the amino-terminal leaderextension is on a light chain of the antibody variant (e.g. on one ortwo light chains of the antibody variant). The main species HER2antibody or the antibody variant may be an full length antibody orantibody fragment (e.g. Fab of F(ab′)2 fragments), but preferably bothare full length antibodies. The antibody variant herein may comprise anamino-terminal leader extension on any one or more of the heavy or lightchains thereof. Preferably, the amino-terminal leader extension is onone or two light chains of the antibody. The amino-terminal leaderextension preferably comprises or consists of VHS—. Presence of theamino-terminal leader extension in the composition can be detected byvarious analytical techniques including, but not limited to, N-terminalsequence analysis, assay for charge heterogeneity (for instance, cationexchange chromatography or capillary zone electrophoresis), massspectrometry, etc. The amount of the antibody variant in the compositiongenerally ranges from an amount that constitutes the detection limit ofany assay (preferably N-terminal sequence analysis) used to detect thevariant to an amount less than the amount of the main species antibody.Generally, about 20% or less (e.g. from about 1% to about 15%, forinstance from 5% to about 15%) of the antibody molecules in thecomposition comprise an amino-terminal leader extension. Such percentageamounts are preferably determined using quantitative N-terminal sequenceanalysis or cation exchange analysis (preferably using ahigh-resolution, weak cation-exchange column, such as a PROPAC WCX-10™cation exchange column). Aside from the amino-terminal leader extensionvariant, further amino acid sequence alterations of the main speciesantibody and/or variant are contemplated, including but not limited toan antibody comprising a C-terminal lysine residue on one or both heavychains thereof, a deamidated antibody variant, etc.

Moreover, the main species antibody or variant may further compriseglycosylation variations, non-limiting examples of which includeantibody comprising a G1 or G2 oligosaccharide structure attached to theFc region thereof, antibody comprising a carbohydrate moiety attached toa light chain thereof (e.g. one or two carbohydrate moieties, such asglucose or galactose, attached to one or two light chains of theantibody, for instance attached to one or more lysine residues),antibody comprising one or two non-glycosylated heavy chains, orantibody comprising a sialidated oligosaccharide attached to one or twoheavy chains thereof etc.

The composition may be recovered from a genetically engineered cellline, e.g. a Chinese Hamster Ovary (CHO) cell line expressing the HER2antibody, or may be prepared by peptide synthesis.

For more information regarding exemplary pertuzumab compositions, seeU.S. Pat. Nos. 7,560,111 and 7,879,325 as well as US 2009/0202546A1.

A commercial formulation of pertuzumab (PERJETA®) contains pertuzumab420 mg/14 mL (30 mg/mL) in the form of a preservative-free solution forIV infusion. In some embodiments, pertuzumab therapy comprisesadministration of an initial loading dose of 840 mg, following byadministration of a flat maintenance dose of 420 mg every three weeks.

F. Methods and Compositions for Diagnostics and Detection

In certain embodiments, any of the anti-HER2 antibodies provided hereinis useful for detecting the presence of HER2 in a biological sample. Theterm “detecting” as used herein encompasses quantitative or qualitativedetection. A “biological sample” comprises, e.g., a cell or tissue(e.g., biopsy material, including cancerous or potentially cancerousbreast tissue).

In one embodiment, an anti-HER2 antibody for use in a method ofdiagnosis or detection is provided. In a further aspect, a method ofdetecting the presence of HER2 in a biological sample is provided. Incertain embodiments, the method comprises contacting the biologicalsample with an anti-HER2 antibody as described herein under conditionspermissive for binding of the anti-HER2 antibody to HER2, and detectingwhether a complex is formed between the anti-HER2 antibody and HER2 inthe biological sample. Such method may be an in vitro or in vivo method.In one embodiment, an anti-HER2 antibody is used to select subjectseligible for therapy with an anti-HER2 antibody, e.g. where HER2 is abiomarker for selection of patients. In a further embodiment, thebiological sample is a cell or tissue.

In a further embodiment, an anti-HER2 antibody is used in vivo todetect, e.g., by in vivo imaging, a HER2-positive cancer in a subject,e.g., for the purposes of diagnosing, prognosing, or staging cancer,determining the appropriate course of therapy, or monitoring response ofa cancer to therapy. One method known in the art for in vivo detectionis immuno-positron emission tomography (immuno-PET), as described, e.g.,in van Dongen et al., The Oncologist 12:1379-1389 (2007) and Verel etal., J. Nucl. Med. 44:1271-1281 (2003). In such embodiments, a method isprovided for detecting a HER2-positive cancer in a subject, the methodcomprising administering a labeled anti-HER2antibody to a subject havingor suspected of having a HER2-positive cancer, and detecting the labeledanti-HER2 antibody in the subject, wherein detection of the labeledanti-HER2 antibody indicates a HER2-positive cancer in the subject. Incertain of such embodiments, the labeled anti-HER2 antibody comprises ananti-HER2 antibody conjugated to a positron emitter, such as ⁶⁸Ga, ¹⁸F,⁶⁴Cu, ⁸⁶Y, ⁷⁶Br, ⁸⁹Zr, and ¹²⁴I. In a particular embodiment, thepositron emitter is ⁸⁹Zr.

In further embodiments, a method of diagnosis or detection comprisescontacting a first anti-HER2 antibody immobilized to a substrate with abiological sample to be tested for the presence of HER2, exposing thesubstrate to a second anti-HER2 antibody, and detecting whether thesecond anti-HER2 is bound to a complex between the first anti-HER2antibody and HER2 in the biological sample. A substrate may be anysupportive medium, e.g., glass, metal, ceramic, polymeric beads, slides,chips, and other substrates. In certain embodiments, a biological samplecomprises a cell or tissue. In certain embodiments, the first or secondanti-HER2 antibody is any of the antibodies described herein.

Exemplary disorders that may be diagnosed or detected according to anyof the above embodiments include HER2-positive cancers, such asHER2-positive breast cancer and HER2-positive gastric cancer. In someembodiments, HER2-positive cancer has an immunohistochemistry (IHC)score of 2+ or 3+ and/or an in situ hybridization (ISH) amplificationratio ≥2.0.

In certain embodiments, labeled anti-HER2 antibodies are provided.Labels include, but are not limited to, labels or moieties that aredetected directly (such as fluorescent, chromophoric, electron-dense,chemiluminescent, and radioactive labels), as well as moieties, such asenzymes or ligands, that are detected indirectly, e.g., through anenzymatic reaction or molecular interaction. Exemplary labels include,but are not limited to, the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I,fluorophores such as rare earth chelates or fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone,luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S.Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase,glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase,galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclicoxidases such as uricase and xanthine oxidase, coupled with an enzymethat employs hydrogen peroxide to oxidize a dye precursor such as HRP,lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,bacteriophage labels, stable free radicals, and the like. In anotherembodiment, a label is a positron emitter. Positron emitters include butare not limited to ⁶⁸Ga, ¹⁸F, ⁶⁴Cu, ⁸⁶Y, ⁷⁶Br, ⁸⁹Zr, and ¹²⁴I. In aparticular embodiment, a positron emitter is ⁸⁹Zr.

G. Pharmaceutical Formulations

Pharmaceutical formulations of an anti-HER2 antibody or immunoconjugateas described herein are prepared by mixing such antibody orimmunoconjugate having the desired degree of purity with one or moreoptional pharmaceutically acceptable carriers (Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the formof lyophilized formulations or aqueous solutions. Pharmaceuticallyacceptable carriers are generally nontoxic to recipients at the dosagesand concentrations employed, and include, but are not limited to:buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as polyethylene glycol (PEG). Exemplarypharmaceutically acceptable carriers herein further includeinsterstitial drug dispersion agents such as soluble neutral-activehyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, BaxterInternational, Inc.). Certain exemplary sHASEGPs and methods of use,including rHuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined withone or more additional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody or immunoconjugate formulations aredescribed in U.S. Pat. No. 6,267,958. Aqueous antibody orimmunoconjugate formulations include those described in U.S. Pat. No.6,171,586 and WO2006/044908, the latter formulations including ahistidine-acetate buffer.

The formulation herein may also contain more than one active ingredientas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody or immunoconjugate, whichmatrices are in the form of shaped articles, e.g. films, ormicrocapsules.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

H. Therapeutic Methods and Compositions

Any of the anti-HER2 antibodies or immunoconjugates provided herein maybe used in methods, e.g., therapeutic methods.

In one aspect, an anti-HER2 antibody or immunoconjugate provided hereinis used in a method of inhibiting proliferation of a HER2-positive cell,the method comprising exposing the cell to the anti-HER2 antibody orimmunoconjugate under conditions permissive for binding of the anti-HER2antibody or immunoconjugate to HER2 on the surface of the cell, therebyinhibiting the proliferation of the cell. In certain embodiments, themethod is an in vitro or an in vivo method. In further embodiments, thecell is a breast cancer cell or a gastric cancer cell.

Inhibition of cell proliferation in vitro may be assayed using theCellTiter-Glo™ Luminescent Cell Viability Assay, which is commerciallyavailable from Promega (Madison, Wis.).

That assay determines the number of viable cells in culture based onquantitation of ATP present, which is an indication of metabolicallyactive cells. See Crouch et al. (1993) J. Immunol. Meth. 160:81-88, U.S.Pat. No. 6,602,677. The assay may be conducted in 96- or 384-wellformat, making it amenable to automated high-throughput screening (HTS).See Cree et al. (1995) AntiCancer Drugs 6:398-404. The assay procedureinvolves adding a single reagent (CellTiter-Glo® Reagent) directly tocultured cells. This results in cell lysis and generation of aluminescent signal produced by a luciferase reaction. The luminescentsignal is proportional to the amount of ATP present, which is directlyproportional to the number of viable cells present in culture. Data canbe recorded by luminometer or CCD camera imaging device. Theluminescence output is expressed as relative light units (RLU).

In another aspect, an anti-HER2 antibody or immunoconjugate for use as amedicament is provided. In further aspects, an anti-HER2 antibody orimmunoconjugate for use in a method of treatment is provided. In certainembodiments, an anti-HER2 antibody or immunoconjugate for use intreating HER2-positive cancer is provided. In certain embodiments, theinvention provides an anti-HER2 antibody or immunoconjugate for use in amethod of treating an individual having a HER2-positive cancer, themethod comprising administering to the individual an effective amount ofthe anti-HER2 antibody or immunoconjugate. In one such embodiment, themethod further comprises administering to the individual an effectiveamount of at least one additional therapeutic agent, e.g., as describedbelow.

In a further aspect, the invention provides for the use of an anti-HER2antibody or immunoconjugate in the manufacture or preparation of amedicament. In one embodiment, the medicament is for treatment ofHER2-positive cancer. In a further embodiment, the medicament is for usein a method of treating HER2-positive cancer, the method comprisingadministering to an individual having HER2-positive cancer an effectiveamount of the medicament. In one such embodiment, the method furthercomprises administering to the individual an effective amount of atleast one additional therapeutic agent, e.g., as described below.

In a further aspect, the invention provides a method for treatingHER2-positive cancer. In one embodiment, the method comprisesadministering to an individual having such HER2-positive cancer aneffective amount of an anti-HER2 antibody or immunoconjugate. In onesuch embodiment, the method further comprises administering to theindividual an effective amount of at least one additional therapeuticagent, as described below.

A HER2-positive cancer according to any of the above embodiments may be,e.g., HER2-positive breast cancer or HER2-positive gastric cancer. Insome embodiments, HER2-positive cancer has an immunohistochemistry (IHC)score of 2+ or 3+ and/or an in situ hybridization (ISH) amplificationratio ≥2.0.

An “individual,” “patient,” or “subject” according to any of the aboveembodiments may be a human.

In a further aspect, the invention provides pharmaceutical formulationscomprising any of the anti-HER2 antibodies or immunoconjugate providedherein, e.g., for use in any of the above therapeutic methods. In oneembodiment, a pharmaceutical formulation comprises any of the anti-HER2antibodies or immunoconjugates provided herein and a pharmaceuticallyacceptable carrier. In another embodiment, a pharmaceutical formulationcomprises any of the anti-HER2 antibodies or immunoconjugates providedherein and at least one additional therapeutic agent, e.g., as describedbelow.

Antibodies or immunoconjugates of the invention can be used either aloneor in combination with other agents in a therapy. For instance, anantibody or immunoconjugate of the invention (e.g., a hu7C2.v.2.2.LAantibody-drug conjugate (hu7C2 ADC)) may be co-administered with atleast one additional therapeutic agent. In some embodiments, theadditional therapeutic agent is also an antibody or immunoconjugate thatbinds to HER2. In some embodiments, the additional therapeutic agent is(i) an antibody or immunoconjugate that binds to domain II of HER2,and/or (ii) an antibody or immunoconjugate that binds to domain IV orHER2. In some embodiments, the additional therapeutic agent is (i) anantibody or immunoconjugate that binds to epitope 2C4, and/or (ii) anantibody or immunoconjugate that binds to epitope 4D5.

In some embodiments, a hu7C2.v.2.2.LA antibody-drug conjugate (hu7C2ADC) is co-administered with one or more additional therapeutic agentsselected from trastuzumab (Herceptin®), T-DM1 (Kadcyla®) and pertuzumab(Perjeta®). In some embodiments, an hu7C2 ADC is co-administered withtrastuzumab. In some embodiments, a hu7C2 ADC is co-administered withT-DM1. In some embodiments, a hu7C2 ADC is co-administered withpertuzumab. In some embodiments, a hu7C2 ADC is co-administered withtrastuzumab and pertuzumab. In some embodiments, a hu7C2 ADC isco-administered with T-DM1 and pertuzumab.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate formulations), and separate administration, in which case,administration of the antibody or immunoconjugate of the invention canoccur prior to, simultaneously, and/or following, administration of theadditional therapeutic agent and/or adjuvant. Antibodies orimmunoconjugates of the invention can also be used in combination withradiation therapy.

An antibody or immunoconjugate of the invention (and any additionaltherapeutic agent) can be administered by any suitable means, includingparenteral, intrapulmonary, and intranasal, and, if desired for localtreatment, intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. Dosing can be by any suitable route, e.g.by injections, such as intravenous or subcutaneous injections, dependingin part on whether the administration is brief or chronic. Variousdosing schedules including but not limited to single or multipleadministrations over various time-points, bolus administration, andpulse infusion are contemplated herein.

Antibodies or immunoconjugates of the invention would be formulated,dosed, and administered in a fashion consistent with good medicalpractice. Factors for consideration in this context include theparticular disorder being treated, the particular mammal being treated,the clinical condition of the individual patient, the cause of thedisorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners. The antibody or immunoconjugate need notbe, but is optionally formulated with one or more agents currently usedto prevent or treat the disorder in question. The effective amount ofsuch other agents depends on the amount of antibody or immunoconjugatepresent in the formulation, the type of disorder or treatment, and otherfactors discussed above. These are generally used in the same dosagesand with administration routes as described herein, or about from 1 to99% of the dosages described herein, or in any dosage and by any routethat is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of anantibody or immunoconjugate of the invention (when used alone or incombination with one or more other additional therapeutic agents) willdepend on the type of disease to be treated, the type of antibody orimmunoconjugate, the severity and course of the disease, whether theantibody or immunoconjugate is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody or immunoconjugate, and the discretion ofthe attending physician. The antibody or immunoconjugate is suitablyadministered to the patient at one time or over a series of treatments.Depending on the type and severity of the disease, about 1 μg/kg to 15mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of antibody or immunoconjugate can be aninitial candidate dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion. One typical daily dosage might range from about 1 μg/kg to 100mg/kg or more, depending on the factors mentioned above. For repeatedadministrations over several days or longer, depending on the condition,the treatment would generally be sustained until a desired suppressionof disease symptoms occurs. One exemplary dosage of the antibody orimmunoconjugate would be in the range from about 0.05 mg/kg to about 10mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kgor 10 mg/kg (or any combination thereof) may be administered to thepatient. Such doses may be administered intermittently, e.g. every weekor every three weeks (e.g. such that the patient receives from about twoto about twenty, or e.g. about six doses of the antibody). An initialhigher loading dose, followed by one or more lower doses may beadministered. However, other dosage regimens may be useful. The progressof this therapy is easily monitored by conventional techniques andassays.

It is understood that any of the above formulations or therapeuticmethods may be carried out using both an immunoconjugate of theinvention and an anti-HER2 antibody.

I. Articles of Manufacture

Articles of manufacture, or “kits”, containing a hu7C2.v.2.2.LAantibody-drug conjugate (hu7C2 ADC) and trastuzumab-MCC-DM1 and/orpertuzumab useful for the treatment methods herein are provided. In someembodiments, the kit comprises a container comprising a hu7C2 ADC. Insome embodiments, the kit further comprises a container comprisingtrastuzumab-MCC-DM1. In some embodiments, the kit further comprisescontainer comprising pertuzumab. In some embodiments, a kit furthercomprises a container comprising trastuzumab-MCC-DM1 and a containercomprising pertuzumab. In some embodiments, the kit comprises two ormore of hu7C2 ADC, trastuzumab-MCC-DM1, and pertuzumab in the samecontainer. The kit may further comprise a label or package insert, on orassociated with the container. The term“package insert” is used to referto instructions customarily included in commercial packages oftherapeutic products, that contain information about the indications,usage, dosage, administration, contraindications and/or warningsconcerning the use of such therapeutic products. Suitable containersinclude, for example, bottles, vials, syringes, blister pack, etc. Thecontainer may be formed from a variety of materials such as glass orplastic. The container may hold hu7C2 ADC and, optionally,trastuzumab-MCC-DM1 and/or pertuzumab or a formulation thereof which iseffective for use in a treatment method herein, and may have a sterileaccess port (for example, the container may be an intravenous solutionbag or a vial having a stopper pierceable by a hypodermic injectionneedle). The label or package insert indicates that the composition isused in a treatment method as described and claimed herein. The articleof manufacture may also contain a further container comprising apharmaceutically acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution anddextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

The kit may further comprise directions for the administration of hu7C2ADC and, optionally, trastuzumab-MCC-DM1 and/or pertuzumab. For example,if the kit comprises a first composition comprising hu7C2 ADC and asecond pharmaceutical formulation, the kit may further comprisedirections for the simultaneous, sequential or separate administrationof the first and second pharmaceutical compositions to a patient in needthereof.

In another embodiment, the kits are suitable for the delivery of solidoral forms of hu7C2 ADC and, optionally, trastuzumab-MCC-DM1 and/orpertuzumab, such as tablets or capsules. Such a kit preferably includesa number of unit dosages. Such kits can include a card having thedosages oriented in the order of their intended use. An example of sucha kit is a “blister pack”. Blister packs are well known in the packagingindustry and are widely used for packaging pharmaceutical unit dosageforms. If desired, a memory aid can be provided, for example in the formof numbers, letters, or other markings or with a calendar insert,designating the days in the treatment schedule in which the dosages canbe administered.

According to one embodiment, a kit may comprise (a) a first containerwith hu7C2 ADC, and optionally, (b) a second container withtrastuzumab-MCC-DM1 contained therein and/or with pertuzumab containedtherein. In some embodiments, a kit may comprise (a) a first containerwith hu7C2 ADC, (b) a second container with trastuzumab-MCC-DM1contained therein, and (c) a third container with pertuzumab containedtherein. In some embodiments, the kit may further comprise a containercomprising a pharmaceutically-acceptable buffer, such as bacteriostaticwater for injection (BWFI), phosphate-buffered saline, Ringer's solutionand dextrose solution. It may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, and syringes.

Where the kit comprises a composition of hu7C2 ADC andtrastuzumab-MCC-DM1 and/or pertuzumab, the kit may comprise a containerfor containing the separate compositions such as a divided bottle or adivided foil packet, however, the separate compositions may also becontained within a single, undivided container. Typically, the kitcomprises directions for the administration of the separate components.The kit form is particularly advantageous when the separate componentsare preferably administered in different dosage forms (e.g., oral andparenteral), are administered at different dosage intervals, or whentitration of the individual components of the combination is desired bythe prescribing physician.

One embodiment of an article of manufacture herein comprises anintravenous (IV) bag containing a stable mixture of a hu7C2 ADC andpertuzumab and/or T-DM1 suitable for administration to a cancer patient.Optionally, the mixture is in saline solution; for example comprisingabout 0.9% NaCl or about 0.45% NaCl. An exemplary IV bag is a polyolefinor polyvinyl chloride infusion bag, e.g. a 250 mL IV bag. According tosome embodiments of the invention, the mixture includes about 420 mg orabout 840 mg of pertuzumab and from about 100 mg to about 160 mg T-DM1.

Optionally, the mixture in the IV bag is stable for up to 24 hours at 5°C. or 30° C. Stability of the mixture can be evaluated by one or moreassays selected from the group consisting of: color, appearance andclarity (CAC), concentration and turbidity analysis, particulateanalysis, size exclusion chromatography (SEC), ion-exchangechromatography (IEC), capillary zone electrophoresis (CZE), imagecapillary isoelectric focusing (iCIEF), and potency assay.

III. EXAMPLES

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Example 1: Humanization of Murine Antibody 7C2

Anti-HER2 murine antibody 7C2 binds to an epitope in domain I of HER2.See, e.g., PCT Publication No. WO 98/17797. This epitope is distinctfrom the epitope bound by trastuzumab, which binds to domain IV of HER2,and the epitope bound by pertuzumab, which binds to domain II of HER2.See FIGS. 3, 16, and 18. By binding domain IV, trastuzumab disruptsligand-independent HER2-HER3 complexes, thereby inhibiting downstreamsignaling (e.g. PI3K/AKT). In contrast, pertuzumab binding to domain IIprevents ligand-driven HER2 interaction with other HER family members(e.g. HER3, HER1 or HER4), thus also preventing downstream signaltransduction.

Binding of MAb 7C2 to domain I does not result in interference oftrastuzumab or pertuzumab binding to domains IV and II, respectively,thereby offering the potential of combining a MAb 7C2 ADC withtrastuzumab, trastuzumab emtansine (T-DM-1), and/or pertuzumab.

Murine antibody 7C2 (7C2.B9, see PCT Publication No. WO 98/17797) washumanized as follows.

A. Materials and Methods

Residue numbers are according to Kabat (Kabat et al., Sequences ofproteins of immunological interest, 5th Ed., Public Health Service,National Institutes of Health, Bethesda, Md. (1991)).

Direct hypervariable region grafts onto the acceptor human consensusframework. Variants constructed during the humanization of 7C2 wereassessed in the form of an IgG. The VL and VH domains from murine 7C2were aligned with the human VL kappa IV (VL_(KIV)) and human VH subgroupI (VH_(I)) consensus sequences. Hypervariable regions (HVR) from themurine 7C2 (7C2.B9) antibody were engineered into VL_(KIV) and VH_(I)acceptor frameworks to generate CDR-graft variants. From the mu7C2 VLdomain, positions 24-34 (L1), 50-56 (L2) and 89-97 (L3) were graftedinto VL_(KI). From the mu7C2 VH domain, positions 26-35 (H1), 50-65 (H2)and 95-102 (H3) were grafted into VH_(I)(FIGS. 1 and 2). The HVRdefinitions are defined by their sequence hypervariability (Wu, T. T. &Kabat, E. A. (1970)), their structural location (Chothia, C. & Lesk, A.M. (1987)) and their involvement in antigen-antibody contacts (MacCallumet al. J. Mol. Biol. 262: 732-745 (1996)). To evaluate framework vernierpositions that might be important, selected vernier positions weremutated back to the murine sequence. The vernier positions includepositions 4 and 49 in VL and positions 37, 67, 69, 71 and 73 in VH.Three different versions of VL sequences and VH sequences weresynthesized (Blue Heron, Bothell, Wash.) and subsequently subcloned intomammalian expression vectors. By combining the different versions of LCwith HC, a total of nine different hu7C2 graft variants (v 1.1, v1.2,v1.3, v2.1, v2.2, v2.3, v3.1, v3.2 and v3.3) were generated.

Affinity maturation library. A monovalent Fab-g3 display phagemid vectorwith 2 open reading frames under control of a single phoA promoter wasused. The first open reading frame consists of the stII signal sequencefused to the VL and CL domains of the acceptor light chain and thesecond consists of the stII signal sequence fused to the VH and CH1domains of the acceptor heavy chain followed by the minor phage coatprotein P3. The HVR graft variant (7C2.v2.1) was generated by Kunkelmutagenesis using separate oligonucleotides for each hypervariableregion, and displayed on phage as a Fab.

To improve affinity, phage libraries containing changes in eachhypervariable region were generated. Sequence diversity was introducedseparately at each position in the hypervariable regions of 7C2.v2.1using Kunkel mutagenesis. Positions in the hypervariable region of7C2.v2.1 were each fully randomized one at a time to all possible 20amino acids using oligonucleotides encoding NNS. A total of 68libraries, each consisting of 20 members, were made having a singleposition located within one of the hypervariable regions of 7C2 fullyrandomized. Libraries with positions in the same hypervariable regionwere pooled to generate a total of six libraries.

Generation of phage libraries. Oligonucleotides designed to introducediversity into each hypervariable region as outlined above werephosphorylated separately in 20 μl reactions containing 660 ng ofoligonucleotide, 50 mM Tris pH 7.5, 10 mM MgCl₂, 1 mM ATP, 20 mM DTT,and 5 U polynucleotide kinase for 1 h at 37° C.

To generate the affinity maturation library, 68 individual Kunkelmutagenesis reactions were performed in a 96-well PCR plate. From thephosphorylated oligonucleotides reactions (above), 2 μl was added to 500ng Kunkel template in 50 mM Tris pH 7.5, 10 mM MgCl₂ in a final volumeof 25 μl. The mixture was annealed at 90° C. for 1 min, 50° C. for 3 minand then cooled on ice. The annealed template was then filled in byadding 0.5 μl 10 mM ATP, 0.5 μl 10 mM dNTPs (10 mM each of dATP, dCTP,dGTP and dTTP), 1 μl 100 mM DTT, 1 μl 10×TM buffer (0.5 M Tris pH 7.5,0.1 M MgCl₂), 80 U T4 ligase, and 4 U T7 polymerase in a total volume of30 μl for 2 h at room temperature. These filled-in and ligated productswere then each transformed into XL 1-blue cells. The librariescontaining positions in the same CDR region were pooled and recovered in10 ml SOC media for 1 hour at 37° C. Carbenacillin (50 μg/ml) andM13/KO7 helper phage (MOI 10) were added. The cultures were incubatedfor another 30 mins at 37° C. and transferred to 500 ml 2YT containing50 μg/ml carbenacillin and 50 μg/ml kanamycin and grown 20 h at 37° C.

Phage Selections. Her2 extracellular domain (Her2 ECD) was biotinylatedthrough free amines using NHS-PEG4-Biotin (Pierce). For biotinylationreactions, a 4-fold molar excess of biotin reagent was used in PBS.Reactions were followed by dialysis in PBS.

Phage were harvested from the cell culture supernatant and suspended inPBS containing 1% BSA. The phage libraries were incubated withbiotinylated Her2 ECD at room temperature and the phage bound tobiotin-Her2 was then captured for 5 min on neutrAvidin (10 μg/ml) thathad been immobilized in PBS on MaxiSorp microtiter plates (Nunc)overnight at 4° C. Microtiter wells were washed extensively with PBScontaining 0.05% Tween 20 (PBST) and bound phage were eluted byincubating the wells with 20 mM HCl, 500 mM KCl for 30 min. Eluted phagewere neutralized with 1 M Tris, pH 7.5 and amplified using XL1-Bluecells and M13/KO7 helper phage and grown overnight at 37° C. in 2YT, 50μg/ml carbenacillin and 50 μg/ml Kanamycin. The titers of phage elutedfrom a target containing well were compared to titers of phage recoveredfrom a non-target containing well to assess enrichment. Selectionstringency was increased by both decreasing concentration ofbiotinylated Her2 ECD (from 5 nM to 0.2 nM) during binding andincreasing the competition time (from 0 to 60 min at room temperature)with 1 μM of unlabeled Her2 ECD in solution.

Surface plasmon resonance assessment of variants. 7C2 variants wereexpressed as IgG by 293 transient transfection. IgG was purified withprotein A affinity chromatography. The affinity of each 7C2 IgG variantfor Her2 was determined by surface plasmon resonance using aBIAcoreT100. Biacore Series S CM5 sensor chips were immobilized withmonoclonal mouse anti-human IgG (Fc) antibody (Human antibody capturekit, GE Healthcare). Serial 3-fold dilutions of each 7C2 variant wereinjected at a flow rate of 30 l/min. Each sample was analyzed with3-minute association and 10-minute dissociation. After each injectionthe chip was regenerated using 3 M MgCl₂. Binding response was correctedby subtracting the RU from a flow cell capturing an irrelevant IgG atsimilar density. A 1:1 Languir model of simultaneous fitting of k_(on)and k_(off) was used for kinetics analysis.

B. Results and Discussion

Humanization of 7C2. The human acceptor frameworks used for humanizationof 7C2 are based on the human VL kappa IV consensus (VL_(KIV)) and thehuman VH_(I) consensus. The VL and VH domains of murine 7C2 were alignedwith the human VL_(KIV) and VH_(I) domains; hypervariable regions wereidentified and grafted into the human acceptor framework to generate7C2.v1.1. The monovalent affinity of 7C2.v1.1 is decreased 2.5-foldrelative to mu7C2.B9 as assessed by SPR (see Table 2).

TABLE 2 Affinity of 7C2 CDR grafted antibodies VL K_(D) (nM) K4 K4.K49K4.L4.K49  VH VH1 v1.1 v2.1 v3.1 (15 nM) (20 nM) (16 nM) VH1.V71 v1.2v2.2 v3.2 (10 nM) (13 nM) (11 nM) VH1.L37.A67.L69.V71.K73 v1.3 v2.3 v3.3(9 nM) (11 nM) (10 nM)

To improve the binding affinity of 7C2.v1.1, positions 4 and 49 in thelight chain and positions 37, 67, 69, 71 and 73 in the heavy chain werechanged to residues found at these positions in mu7C2.B9. Combinationsof these altered light and heavy chains with chains from 7C2.v1.1 weretransfected into 293 cells, expressed as IgG and purified, and assessedfor binding to Her2 ECD by SPR (see Table 2). Variant 7C2.v3.3, whichcontains 2 altered positions in light chain and 5 altered positions inheavy chain, had a monovalent affinity comparable to mu7C2.B9 (see Table4).

Affinity maturation libraries were explored in an effort to recruitfurther improvements using the framework of 7C2.v2.1, which containsminimal altered vernier position (Y49K) in light chain. For eachhypervariable region, all 20 amino acids were introduced separately atindividual position using Kunkle mutagenesis (a total of 68 libraries,each containing 20 members, pooled into six affinity maturationlibraries). The six affinity maturation libraries were panned for 4rounds in solution with biotinylated Her2 ECD. Selection stringency wasgradually increased by decreasing the concentration of biotin-Her2 ECD(from 5 to 0.2 nM) and increasing the competition time (from 0 to 1 hourat room temperature) with saturated amount of unlabeled Her2 ECD. A twothousand fold of phage enrichment was observed for the H2 library.

A total of 588 clones from the last round were picked for DNA sequenceanalysis. Individual sequence changes were identified in each HVR (seeTable 3). The most abundant clones had changes in VH at position S53 toMet or Leu. The S53M and S53L variants were expressed as IgG and SPRanalysis indicate that S53M and S53L have comparable affinity to Her2.The S53L variant was selected since methionine is prone to oxidationduring the manufacturing process. A potential iso-aspartic acid formingsite in HVR-H2 was eliminated with a S55A mutation (see Table 4).

TABLE 3 Kinetics of affinity-improved variants k_(a) k_(d) K_(D) VariantHVR-Hl HVR-H2 HVR-H3 (1/Ms) (1/s) (nM) v2.1 GYWMN MIHPSDSEIRANQKFRDGTYDGGFEY 2.6E+05 4.1E−03 15.5 (SEQ ID (SEQ ID NO: 8) (SEQ ID NO:NO: 15) 17) v2.1.S53M MIHPMDSEIRANQKFRD 2.7E+05 6.7E−04 2.4(SEQ ID NO: 20) v2.1.S53L MIHPLDSEIRANQKFRD 2.5E+05 8.5E−04 3.4(SEQ ID NO: 21) v2.1.E101K GTYDGGFKY 2.2E+05 1.5E−03 6.8 (SEQ ID NO: 22)

TABLE 4 Summary of hu7C2 variant affinities hu7C2 variant K_(D) (nM)mu7C2.B9 8 hu7C2.v2.2 11 hu7C2.v2.2.LA (S53L, S55A); 3 HVR-H2 of SEQ IDNO: 16

An alignment of the human VL_(KIV) and VH_(I) domains and the heavychain and light chain variable regions of mu7C2.B9 (“7C2”) andhu7C2.v2.2.LA (referred to in the following examples as “hu7C2”) isshown in FIGS. 1 and 2.

Example 2: Production of hu7C2 Antibody Drug Conjugates

For larger scale antibody production, antibodies were produced in CHOcells. Vectors coding for heavy chain and light chain were transfectedinto CHO cells and IgG was purified from cell culture media by protein Aaffinity chromatography.

A. Synthesis of Center-Linked PBD Linker Drug Intermediate

The center-linked PBD linker drug intermediate(N-(3-(3,5-bis((((S)-7-methoxy-2-methylene-5-oxo-2,3,5,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)methyl)phenyl)prop-2-yn-1-yl)-1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3,6,9,12-tetraoxapentadecan-15-amide;“10”) having the following formula:

was synthesized as follows.

1. (11S,11aS,11′S,11a′S)-di-tert-butyl8,8′-(((5-iodo-1,3-phenylene)bis(methylene))bis(oxy))bis(7-methoxy-2-methylene-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate)(2a)

1,3-bis(bromomethyl)-5-iodobenzene (2.00 g, 5.20 mmol) was added to astirred solution of Boc/THP-protected PBD capping unit 1 (4.75 g, 10.3mmol), TBAI (190 mg, 0.52 mmol) and K₂CO₃ (1.42 g, 10.3 mmol) in dry DMF(60 mL). The reaction mixture was heated to 60° C. and stirred under anargon atmosphere for 3 hours at which point analysis by LC/MS revealedsubstantial product formation at retention time 4.15 min (ES+) m/z 1171([M+Na]⁺, ˜10% relative intensity). The reaction mixture was allowed tocool to room temperature and the DMF was removed by evaporation invacuo. The resulting residue was partitioned between water (50 mL) andEtOAc (50 mL) and the aqueous phase was extracted with EtOAc (3×20 mL).The combined organic layers were washed with water (2×20 mL), brine (50mL), dried (MgSO₄), filtered and evaporated in vacuo to provide thecrude product. Purification by flash chromatography (gradient elution:50:50 v/v EtOAc/hexane to 80:20 v/v EtOAc/hexane) gave the bis-ether 2aas a white foam (5.42 g, 91% yield).

2. (11S,11aS,11′S,11a′S)-di-tert-butyl8,8′-(((5-bromo-1,3-phenylene)bis(methylene))bis(oxy))bis(7-methoxy-2-methylene-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate)(2b)

1-bromo-3,5-bis(bromomethyl)benzene (1.54 g, 4.53 mmol) was added to astirred solution of Boc/THP-protected PBD capping unit 1 (4.20 g, 9.06mmol), TBAI (167 mg, 0.45 mmol) and K₂CO₃ (1.25 g, 9.06 mmol) in dry DMF(52 mL). The reaction mixture was heated to 60° C. and stirred under anargon atmosphere for 5 hours at which point analysis by LC/MS revealedsubstantial product formation at retention time 4.10 min (ES+) m/z 1101([M+H]⁺, ˜70% relative intensity). The reaction mixture was allowed tocool to room temperature and the DMF was removed by evaporation invacuo. The resulting residue was partitioned between water (60 mL) andEtOAc (60 mL) and the aqueous phase was extracted with EtOAc (3×25 mL).The combined organic layers were washed with water (30 mL), brine (50mL), dried (MgSO₄), filtered and evaporated in vacuo to provide thecrude product. Purification by flash chromatography (gradient elution:50:50 v/v EtOAc/hexane to 100% EtOAc) gave the bis-ether 2b as a whitefoam (3.37 g, 68% yield).

3. (11S,11aS,11′S,11a′S)-di-tert-butyl8,8′-(((5-chloro-1,3-phenylene)bis(methylene))bis(oxy))bis(7-methoxy-2-methylene-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate)(2c)

1,3-bis(bromomethyl)-5-chlorobenzene (1.42 g, 4.80 mmol) was added to astirred solution of Boc/THP-protected PBD capping unit 1 (4.42 g, 9.60mmol), TBAI (177 mg, 0.48 mmol) and K₂CO₃ (1.33 g, 9.60 mmol) in dry DMF(55 mL). The reaction mixture was heated to 60° C. and stirred under anargon atmosphere for 1.5 hours at which point analysis by LC/MS revealedsubstantial product formation at retention time 4.08 min (ES+) m/z 1057([M+H]⁺, ˜30% relative intensity). The reaction mixture was allowed tocool to room temperature and the DMF was removed by evaporation invacuo. The resulting residue was partitioned between water (60 mL) andEtOAc (60 mL) and the aqueous phase was extracted with EtOAc (3×20 mL).The combined organic layers were washed with water (20 mL), brine (40mL), dried (MgSO₄), filtered and evaporated in vacuo to provide thecrude product. Purification by flash chromatography (gradient elution:50:50 v/v EtOAc/hexane to 80:20 v/v EtOAc/hexane) gave the bis-ether 2cas a white foam (5.10 g, 99% yield).

A catalytic amount of Pd(PPh₃)₄(5.0 mg, 4.2 μmol) was added to a mixtureof the bis-ether 2a (242 mg, 0.21 mmol), propargylamine (41 μL, 35 mg,0.63 mmol), CuI (1.6 mg, 8.4 μmol), diethylamine (0.42 mL, 309 mg, 4.22mmol) and oven-dried 4 Å molecular sieve pellets in dry DMF (1.8 mL) inan oven-dried sealable vessel. The mixture was degased and flushed withargon 3 times then heated in a microwave at 100° C. for 3 minutes atwhich point analysis by LC/MS revealed complete consumption of startingmaterial and substantial product formation at retention time 3.18 min(ES+) m/z 1076 ([M+H]⁺, ˜60% relative intensity). The reaction mixturewas allowed to cool to room temperature and was then filtered through asinter to remove the sieves (washed with DMF). The filtrate wasevaporated in vacuo to provide the unstable crude product 8 which wasused immediately in the next step without purification or analysis.

MAL-dPEG®4-acid (88 mg, 0.21 mmol) was added to a stirred solution ofEDCI (41 mg, 0.21 mmol) and the crude primary amine 8 in dry DCM (4 mL)at room temperature. The reaction mixture was stirred under an argonatmosphere for 3 hours at which point analysis by LC/MS showed asubstantial amount of desired product at retention time 3.58 min (ES+)m/z 1475 ([M+H]⁺, ˜10% relative intensity), 1498 ([M+Na]⁺, ˜5% relativeintensity) accompanied by a side product at retention time 3.85 min. Thereaction mixture was diluted with DCM (30 mL) and washed with H₂O (3×10mL), brine (20 mL), dried (MgSO₄), filtered and evaporated in vacuo toprovide the crude product. Purification by flash chromatography(gradient elution: 100% DCM to 96:4 v/v DCM/MeOH) gave the maleimide 9as a foam (67 mg, 22% yield over 2 steps).

A solution of 95:5 v/v TFA/H₂O (1 mL) was added to a sample of theBoc/THP-protected compound 9 (67 mg, 45.5 μmol) at 0° C. (ice/acetone).After stirring at 0° C. for 1.5 hours, the reaction was deemed completeas judged by LC/MS, desired product peak at retention time 2.67 min(ES+) m/z 1070 ([M+H]⁺, ˜5% relative intensity). The reaction mixturewas kept cold and added drop wise to a chilled saturated aqueoussolution of NaHCO₃ (50 mL). The mixture was extracted with DCM (3×15 mL)and the combined organic layers washed with brine (40 mL), dried(MgSO₄), filtered and evaporated in vacuo to provide the crude product.Purification by flash chromatography (gradient elution: 100% CHCl₃ to96:4 v/v CHCl₃/MeOH) gave 10 as an orange foam (12 mg, 24% yield).

B. Alternate Synthesis of Center-Linked PBD Linker Drug Intermediate

The center-linked PBD linker drug intermediate(N-(3-(3,5-bis((((S)-7-methoxy-2-methylene-5-oxo-2,3,5,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)methyl)phenyl)prop-2-yn-1-yl)-1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3,6,9,12-tetraoxapentadecan-15-amide;“10”) having the following formula:

may also be synthesized as follows.

EDCI (263 mg, 1.37 mmol) was added to a stirred solution oft-boc-N-amido-dPEG®₄-acid (26) (500 mg, 1.37 mmol, Stratech ScientificLimited) and propargylamine (88 μL, 76 mg, 1.37 mmol) in dry DCM (10 mL)at room temperature. The reaction mixture was stirred under an argonatmosphere for 16 hours at which point analysis by LC/MS showed asubstantial amount of desired product at retention time 1.26 minutes(ES+) m/z 403 ([M+H]⁺, ˜50% relative intensity), 425 ([M+Na]⁺, ˜100%relative intensity), note that both starting material and product hadweak UV absorption (214 and 254 nm) and were best detected on ES+TIC.The reaction mixture was diluted with DCM (100 mL) and washed with H₂O(30 mL), brine (40 mL), dried (MgSO₄), filtered and evaporated in vacuoto provide the crude product. Purification by flash chromatography(gradient elution in 1% increments: 100% DCM to 98:2 v/v DCM/MeOH) gavethe amide 27 as an oil (392 mg, 71% yield).

A catalytic amount of Pd(PPh₃)₄(23.0 mg, 19.5 μmol) was added to amixture of the iodoaryl compound 2a (1.02 g, 0.89 mmol), Boc-acetylene27 (393 mg, 0.98 mmol), CuI (7.4 mg, 39.1 μmol), diethylamine (2.02 mL,1.43 g, 19.5 mmol) and oven-dried 4 Å molecular sieve pellets in dry DMF(9 mL) in an oven-dried sealable vessel. The mixture was degased andflushed with argon 3 times then heated in a microwave at 100° C. for 26minutes at which point analysis by LC/MS revealed substantial productformation at retention time 1.89 minutes (ES+) m/z 1446 ([M+Na]⁺, ˜100%relative intensity, 1424 ([M+H]⁺, ˜15% relative intensity). The reactionmixture was allowed to cool to room temperature and was then filteredthrough a sinter to remove the sieves (washed with DMF). The filtratewas evaporated in vacuo and the resulting residue dissolved in DCM (100mL) and washed with H₂O (20 mL), brine (30 mL), dried (MgSO₄), filteredand evaporated in vacuo to give the crude product. Purification by flashchromatography (gradient elution in 1% increments: 100% DCM to 97:3 v/vDCM/MeOH) provided the alkyne 28 as a yellow foam (882 mg, 70% yield).

TBDMSOTf (1.42 mL, 1.64 g, 6.2 mmol) was added to a stirred solution ofthe tri-Boc protected compound 28 (882 mg, 0.62 mmol) and 2,6-lutidine(0.96 mL, 883 mg, 8.25 mmol) in dry DCM (15 mL) at room temperature. Thereaction mixture was allowed to stir under an argon atmosphere for 16hours during which time analysis by LC/MS revealed formation of the TBScarbamate at retention time 2.09 minutes (ES+) m/z 1504 ([M+Na]⁺, ˜100%relative intensity). The reaction mixture was diluted with DCM (60 mL)and washed with saturated NH₄Cl (2×20 mL), H₂O (20 mL), brine (30 mL),dried (MgSO₄), filtered and evaporated in vacuo to give the crude TBScarbamate. The product was re-dissolved in THF (15 mL) and treated witha solution of TBAF (744 μL of a 1.0M solution in THF, 0.744 mmol) atroom temperature. The reaction mixture was allowed to stir for 1 hour atroom temperature at which point analysis by LC/MS revealed substantialproduct formation at retention time 1.45 minutes (ES+) m/z 1324 ([M+H]⁺,˜60% relative intensity) along with product corresponding to 1 N10Boc/1THP cleaved at retention time 1.29 minutes (ES+) m/z 1121 ([M+H]⁺, ˜10%relative intensity), 1138 ([M+H₂O]⁺, ˜20% relative intensity) andproduct corresponding to 2 N10 Boc/2 THP cleaved at retention time 1.12minutes (ES+) m/z 919 ([M+H]⁺, ˜2.5% relative intensity), 937 ([M+H₂O]⁺,˜3% relative intensity), 955 ([M+2H₂O]⁺, ˜5% relative intensity). TheTHF was removed by evaporation in vacuo and the resulting residuere-dissolved in DCM (60 mL) and washed with saturated NH₄Cl (2×20 mL),H₂O (20 mL), brine (30 mL), dried (MgSO₄), filtered and evaporated invacuo to give the key amine 29 as a pinkish foam.

EDCI (61 mg, 0.32 mmol) was added to a stirred solution ofN-maleoyl-β-alanine (53 mg, 0.32 mmol) and amine 29 (˜418 mg, 0.32 mmol)in dry DCM (6 mL) at room temperature. The reaction mixture was stirredunder an argon atmosphere for 3 hours at which point analysis by LC/MSshowed a substantial amount of desired product at retention time 1.80minutes (ES+) m/z 1474 ([M+H]⁺, ˜15% relative intensity), 1497 ([M+Na]⁺,˜100% relative intensity), along with product corresponding to 1N10Boc/1 THP cleaved at retention time 1.56 minutes 1272 ([M+H]⁺, ˜80%relative intensity), 1295 ([M+Na]⁺, ˜45% relative intensity) and productcorresponding to 2 N10 Boc/2 THP cleaved at retention time 1.31 minutes(ES+M+ not observed). The reaction mixture was diluted with DCM (30 mL)and washed with H₂O (15 mL), brine (20 mL), dried (MgSO₄), filtered andevaporated in vacuo to provide the crude product 9 as a foam.

A solution of 95:5 v/v TFA/H₂O (5 mL) was added to a crude sample of theBoc/THP-protected compound 9 (˜466 mg, 0.32 mmol) at 0° C.(ice/acetone). After stirring at 0° C. for 1 hour the reaction wasdeemed complete as judged by LC/MS, desired product peak at retentiontime 1.32 minutes (ES+) m/z 1070 ([M+H]⁺, ˜100% relative intensity). Thereaction mixture was kept cold and added drop-wise to a chilledsaturated aqueous solution of NaHCO₃ (120 mL). The mixture was extractedwith DCM (3×40 mL) and the combined organic layers washed with brine (50mL), dried (MgSO₄), filtered and evaporated in vacuo to provide thecrude product. Purification by flash chromatography (gradient elution:100% CHCl₃ to 96:4 v/v CHCl₃/MeOH) gave 10 as an orange foam (202 mg,60% yield): [α]²¹ _(D)=+351° (c=0.47, CHCl₃); LC/MS (15-minute run),retention time 4.88 minutes (ES+) m/z 1070 ([M+H]⁺, ˜100% relativeintensity); ¹H NMR (400 MHz, CDCl₃) δ 7.66 (d, 2H, J=4.4 Hz), 7.52 (s,2H), 7.45-7.40 (m, 3H), 6.98-6.94 (m, 1H), 6.80 (s, 2H), 6.66 (s, 2H),6.55-6.50 (m, 1H), 5.22-5.07 (m, 8H), 4.30-4.22 (m, 6H), 3.96 (s, 6H),3.91-3.85 (m, 2H), 3.82 (t, 2H, J=7.2 Hz), 3.76 (t, 2H, J=5.8 Hz),3.65-3.43 (m, 16H), 3.16-3.08 (m, 2H), 2.94 (d, 2H, J=15.7 Hz),2.54-2.44 (m, 4H).

C. Conjugation of Linker-Drug Moieties to Antibodies

Hu7C2 antibody-drug conjugates (ADCs) are produced by conjugatinghu7C2.v.2.2.LA with a heavy chain A118C mutation (thio-hu7C2-HC A118C)or a light chain K149C mutation (thio-hu7C2-LC-K149C) to the selecteddrug-linker moiety (e.g., center-linked PBD linker-drug intermediate).As initially isolated, the engineered cysteine residues in antibodiesexist as mixed disulfides with cellular thiols (e.g., glutathione) andare thus unavailable for conjugation. Partial reduction of theseantibodies (e.g., with DTT), purification, and reoxidation withdehydroascorbic acid (DHAA) gives antibodies with free cysteinesulfhydryl groups available for conjugation, as previously described,e.g., in Junutula et al. (2008) Nat. Biotechnol. 26:925-932 and US2011/0301334. Briefly, the antibodies are combined with the drug-linkermoiety to allow conjugation of the drug-linker moiety to the freecysteine residues of the antibody. After several hours, the ADCs arepurified. The drug load (average number of drug moieties per antibody)for the ADC was determined and was in the range of 1.8-1.9. Theresulting ADC structures and the terms used for them below are shown inFIG. 6.

An example of a method for conjugation of linker-drug moieties toantibodies is as follows:

Full length, cysteine engineered monoclonal antibodies(THIOMABS™-Junutula, et al., 2008b Nature Biotech., 26(8):925-932;Dornan et al (2009) Blood 114(13):2721-2729; U.S. Pat. Nos. 7,521,541;7,723,485; WO2009/052249, Shen et al (2012) Nature Biotech., 30(2):184-191; Junutula et al (2008) Jour of Immun. Methods 332:41-52)expressed in CHO cells are reduced with about a 20-40 fold excess ofTCEP (tris(2-carboxyethyl)phosphine hydrochloride or DTT(dithiothreitol) in 50 mM Tris pH 7.5 with 2 mM EDTA for 3 hrs at 37° C.or overnight at room temperature. (Getz et al (1999) Anal. Biochem.273:73-80; Soltec Ventures, Beverly, Mass.). The reduced THIOMAB™ isdiluted and loaded onto a HiTrap S column in 10 mM sodium acetate, pH 5,and eluted with PBS containing 0.3M sodium chloride. Alternatively, theantibody is acidified by addition of 1/20th volume of 10% acetic acid,diluted with 10 mM succinate pH 5, loaded onto the column and thenwashed with 10 column volumes of succinate buffer. The column is elutedwith 50 mM Tris pH7.5, 2 mM EDTA.

The eluted reduced THIOMAB™ is treated with 15 fold molar excess of DHAA(dehydroascorbic acid) or 200 nM aqueous copper sulfate (CuSO4).Oxidation of the interchain disulfide bonds is complete in about threehours or more. Ambient air oxidation is also effective. The re-oxidizedantibody is dialyzed into 20 mM sodium succinate pH 5, 150 mM NaCl, 2 mMEDTA and stored frozen at −20° C.

The deblocked, reoxidized, thio-antibodies (THIOMABS™) are reacted with6-8 fold molar excess of the selected drug-linker moiety (e.g.,center-linked PBD linker-drug intermediate) (from a DMSO stock at aconcentration of 20 mM) in 50 mM Tris, pH 8, until the reaction iscomplete (16-24 hours) as determined by LC-MS analysis of the reactionmixture.

The crude antibody-drug conjugates (ADC) are then applied to a cationexchange column after dilution with 20 mM sodium succinate, pH 5. Thecolumn is washed with at least 10 column volumes of 20 mM sodiumsuccinate, pH 5, and the antibody is eluted with PBS. The antibody drugconjugates are formulated into 20 mM His/acetate, pH 5, with 240 mMsucrose using gel filtration columns. The antibody-drug conjugates arecharacterized by UV spectroscopy to determine protein concentration,analytical SEC (size-exclusion chromatography) for aggregation analysisand LC-MS before and after treatment with Lysine C endopeptidase.

Size exclusion chromatography is performed using a Shodex KW802.5 columnin 0.2M potassium phosphate pH 6.2 with 0.25 mM potassium chloride and15% IPA at a flow rate of 0.75 ml/min. Aggregation state of theconjugate is determined by integration of eluted peak area absorbance at280 nm.

LC-MS analysis is performed using an Agilent QTOF 6520 ESI instrument.As an example, an antibody-drug conjugate generated using this chemistryis treated with 1:500 w/w Endoproteinase Lys C (Promega) in Tris, pH7.5, for 30 min at 37° C. The resulting cleavage fragments are loadedonto a 1000A, 8 um PLRP-S column heated to 80° C. and eluted with agradient of 30% B to 40% B in 5 minutes. Mobile phase A is H₂O with0.05% TFA and mobile phase B is acetonitrile with 0.04% TFA. The flowrate is 0.5 ml/min. Protein elution is monitored by UV absorbancedetection at 280 nm prior to electrospray ionization and MS analysis.Chromatographic resolution of the unconjugated Fc fragment, residualunconjugated Fab and drugged Fab is usually achieved. The obtained m/zspectra were deconvoluted using Mass Hunter™ software (AgilentTechnologies) to calculate the mass of the antibody fragments.

Example 3: Efficacy of hu7C2 Antibody Drug Conjugates in MMTV-Her2 Fo5Transgenic Mammary Tumor Transplant Xenograft Models

CRL nu/nu mice (Charles River Laboratory) were implanted with ˜2×2 mmfragments of MMTV-Her2 Fo5 transgenic breast tumors. When tumors reacheda mean tumor volume of 100-300 mm³, animals were grouped into 7 groupsof 8 mice each. The mice received a single administration on day 1 ofone of the following treatments, via intravenous tail vein injection:(1) vehicle (20 mM L-histidine, 240 mM sucrose, 0.02% Tween-20, pH 5.5),(2) thio-hu7C2-HC-A118C-center-linked-PBD, 0.3 mg/kg; (3)thio-hu7C2-HC-A118C-center-linked-PBD, 1 mg/kg; (4) thio-hu7C2-LC-K149C-center-linked-PBD, 0.3 mg/kg; (5) thio-hu7C2-LC-K149C-center-linked-PBD, 1 mg/kg; (6)thio-controlAb-HC-A118C-center-linked-PBD, 1 mg/kg; or (7)thio-controlAb-LC-K149C-center-linked-PBD, 1 mg/kg. The DAR for thethio-hu7C2-LC-K149C-center-linked-PBD, the thio-hu7C2-HC-A18C-center-linked-PBD, and the thio-controlAb-LC-K149C-center-linked-PBDwas 1.8. The DAR for the thio-controlAb-HC-A118C-center-linked-PBD was1.9.

Tumor and body weight measurements were taken at least once per week forthe duration of the study. Mice were euthanized when tumors reached1000-2000 mm³ or if the mouse lost 20% or more of its body weight. Tumorvolume was measured in two dimensions (length and width) using calipersand the tumor volume was calculated using the formula: Tumor size(mm³)=(longer measurement×shorter measurement²)×0.5.

The results of that experiment are shown in Table 5 and FIG. 4. Eachgroup contained 8 mice at the beginning of the study. AUC/day % TGI(tumor growth inhibition) is calculated using the following formula: %TGI=100×(1−AUCtreatment/Day÷AUCvehicle/Day). PR=partial response, whichis defined as more than 50% but less than 100% reduction in tumorvolume, compared with the starting tumor volume, on any day during thestudy. CR=complete response, which is defined as a 100% reduction intumor volume (no measurable tumor), on any day during the study.

TABLE 5 Efficacy of hu7C2 ADCs in MMTV-Her2 Fo5 transgenic mammary tumorxenograft model tumor Last % BW volume, Day AUC/day % TGI change, lastGroup last day (N) (lower, upper) PR CR day (1) vehicle 1640 14 (7) 0(0, 0)  0 0 5.71 (2) thio-hu7C2-HC- 260 35 (6)  93 (77, 103) 1 0 2.18A118C-center-linked- PBD, 0.3 mg/kg (3) thio-hu7C2-HC- 115 43 (6) 106(95, 118) 2 2 5.83 A118C-center-linked- PBD, 1 mg/kg (4) thio-hu7C2-LC-194 43 (6)  97 (82, 107) 2 0 5.15 K149C-center-linked- PBD, 0.3 mg/kg(5) thio-hu7C2-LC- 67 43 (8)  111 (101, 123) 5 3 3.53K149C-center-linked- PBD, 1 mg/kg (6) thio-controlAb-HC- 794 22 (8) 54(10, 79) 0 0 7.66 A118C-center-linked- PBD, 1 mg/kg (7)thio-controlAb-LC- 512 26 (6) 72 (39, 89) 0 0 4.62 K149C-center-linked-PBD, 1 mg/kg

As shown in Table 5, thio-hu7C2-LC-K149C-center-linked-PBD showed 5partial responses and 3 complete responses at 1 mg/kg and 2 partialresponses at 0.3 mg/kg. Thio-hu7C2-HC-A118C-disulfide-PBD showed 2partial responses and 2 complete responses at 1 mg/kg and 1 partialresponse at 0.3 mg/kg.

Example 4: Efficacy of hu7C2 Antibody Drug Conjugates in KPL-4 HumanBreast Cancer Transgenic Mammary Tumor Transplant Xenograft Models

Female C.B-17 SCID-beige mice (Charles River Laboratory) were eachinoculated in the thoracic mammary fat pad area with 3 million KPL-4cells suspended in HBSS/matrigel (1:1 ratio). When the xenograft tumorsreached an average tumor volume of 100-300 mm3 (referred to as Day 0),animals were randomized into 5 groups of 8 mice each. The mice receiveda single administration on day 1 of one of the following treatments, viaintravenous tail vein injection: (1) vehicle (20 mM L-histidine, 240 mMsucrose, 0.02% Tween-20, pH 5.5), (2)thio-hu7C2-LC-K149C-center-linked-PBD, 0.3 mg/kg; (3)thio-hu7C2-LC-K149C-center-linked-PBD, 1 mg/kg; (4)thio-hu7C2-LC-K149C-center-linked-PBD, 3 mg/kg; (5)thio-controlAb-LC-K149C-center-linked-PBD, 3 mg/kg. The DAR for thethio-hu7C2-LC-K149C-center-linked-PBD was 1.7 and the DAR for thecontrol was 1.8.

Tumors and body weights of mice were measured 1-2 times a weekthroughout the study. Mice were promptly euthanized when body weightloss was >20% of their starting weight. Tumor volume was measured in twodimensions (length and width) using calipers and the tumor volume wascalculated using the formula: Tumor size (mm3)=(longermeasurement×shorter measurement 2)×0.5.

The results of that experiment are shown in Table 6 and FIG. 5. Eachgroup contained 8 mice at the beginning of the study. AUC/day % TGI(tumor growth inhibition) is calculated using the following formula: %TGI=100×(1−AUCtreatment/Day÷AUCvehicle/Day). PR=partial response, whichis defined as more than 50% but less than 100% reduction in tumorvolume, compared with the starting tumor volume, on any day during thestudy. CR=complete response, which is defined as a 100% reduction intumor volume (no measurable tumor), on any day during the study.

TABLE 6 Efficacy of hu7C2 ADCs in KPL-4 Human Breast Cancer TransgenicMammary Tumor Transplant Xenograft Models tumor Last AUC/day % % BWvolume, last Day TGI change, last Group day (N) (lower, upper) PR CR day(1) vehicle 1095 22 (8)  0 (0, 0) 0 0 −5.89 (2) thio-hu7C2-HC- 550 25(8)  58 (−14, 87) 0 0 −2.45 A149C-center-linked- PBD, 0.3 mg/kg (3)thio-hu7C2-HC- 641 25 (7)  55 (−28, 90) 0 0 −5.7 A149C-center-linked-PBD, 1 mg/kg (4) thio-hu7C2-HC- 79 25 (8) 120 (108, 147) 5 1 −5.94A149C-center-linked- PBD, 3 mg/kg (5) thio-controlAb-LC- 247 25 (6) 107(87, 128) 1 0 −14.99 K149C-center-linked- PBD, 3 mg/kg

As shown in Table 6, thio-hu7C2-LC-K149C-center-linked-PBD showed 5partial responses and 1 complete response at 1 mg/kg.

In this study, anti-Her2 ADC demonstrated dose-dependent inhibition oftumor growth, resulting in tumor regression at 3 mg/kg dose.

Example 3: Crystal Structure of 7C2 Fab Bound to HER2 Methods

Expression, purification, and crystallization of the 7C2/HER2complex—7C2 Fab was expressed in E. coli and purified using Protein Gsepharose affinity resin (GE), SP sepharose cation exchangechromatography, and size exclusion chromatography (SEC). HER2extracellular domain (ECD) was expressed in CHO cells and purified byaffinity chromatography using trastuzumab antibody linked to controlledpore glass beads, followed by DEAE anion exchange and size exclusionchromatography.

The complex between Fab 7C2 and HER2 ECD was purified by SEC. Thecomplex was deglycosylated using a combination of enzymes (Endo F1, F2,F3, Endo H and PNGase), followed by purification by SEC into 0.1M NaCl,20 mM HEPES pH 7.2 and 2% glycerol. The complex was crystallizedresulting in thick plates after one week in hanging drops using equalparts of protein at 10 mg/mL and reservoir (30% v/v PEG 550monomethylether, 0.1M Sodium citrate tribasic dihydrate pH 5.0) andtreated briefly with reservoir prior to immersion in liquid nitrogen.

The diffraction data for the complex extending to 2.7 Å resolution werecollected at ˜110 K at SSRL beam line 11-1. The diffraction images wereintegrated and scaled using the program HKL2000 and elements of the CCP4suite. See Winn et al., 2011, Acta Crystallogr D. Biol. Crystallogr. 67:235-42.

The structure was solved by molecular replacement (MR) using programPhaser. See McCoy et al., 2005, Acta Crystallogr D. Biol. Crystallogr.61: 458-64. The MR search models include the HER2 ECD domain derivedfrom a crystal structure of HER2/Herceptin Fab complex (PDB code: 1N8Z),Fab constant domain (PDB code: 1N8Z) and a predicted model for thevariable domain generated by the program Modeller. See Fiser et al.,2003, Methods Enzymol., 374: 461-91. The structure was refined withprograms REFMAC5 (Marshudov et al., 2011, Acta Crystallogr D. Biol.Crystallogr. 67: 355-67) and PHENIX.refine (Adams et al., 2010, ActaCrystallogr D. Biol. Crystallogr. 66 (pt. 2): 213-21) using the maximumlikelihood target functions, anisotropic individual B-factors, and TLSrefinement. The data and refinement statistics are summarized in Table7.

TABLE 7 Statistics of x-ray diffraction data collection and structurerefinement (values in parentheses are for last resolution shell) Datacollection SSRL 11-1 Space group C222₁ Cell parameters (Å) a = 136.8, b= 171.9, c = 162.5 Resolution (Å) 50-2.75 (2.85-2.75) Rsym 0.112 (0.689)Number of observations 319169 Unique reflections 49078 Redundancy 6.5(5.5) Completeness (%) 98.8 (92.2) <I>/<σI> 20 (2.2) Vm(A³/Da) 4.2Refinement Resolution (Å) 48.33-2.75 Number of reflections 49053 R,Rfree 0.23, 0,25 Number of residues 1047 Number of waters 109 Number ofatoms 8039 RMSD bonds (Å) 0.007 RMSD angles (″) 1.2 Mean bonded ΔB (Å²)5.5 Ramachandran analysis (%) 93/6/1 Number of TLS groups 3 <B>^(e) (Å²)7C2/HER2 88

Results

The crystal structure of the 7C2 Fab/HER2 complex was determined at 2.75Å resolution. Each asymmetric unit cell contains one Fab/HER2 complex.The structure revealed that the 7C2 Fab binds to domain I of HER2 ECD(FIG. 11A). The binding epitope is distinct from those in the previouslycharacterized complexes of HER2 ECD with Fab fragments of therapeuticantibodies trastuzumab (Tmab) or pertuzumab (Pmab), which are located atdomains IV and II, respectively. See, e.g., Cho et al., 2003, Nature,421: 756-60; Eigenbrot et al., 2010, PNAS, 107: 15039-44; and Franklinet al., 2004, Caner Cell, 5: 17-28. Indeed, an overlay of the 7C2Fab/HER2 ECD complex structure with the structures of Tmab/HER2 ECDcomplex and Pmab/HER2 ECD complex shows that the three Fabs haveindependent, non-overlapping epitopes and would not spatially interferewith each other binding to HER2 (FIG. 11A). A superposition of the HER2ECD structures within the Tmab/HER2 ECD complex, Pmab/HER2 ECD complexand 7C2 Fab/HER2 ECD complex showed a minimal structural differences(FIG. 11B). This observation suggested that the HER2 ECD is relativelyrigid, which is consistent with previous reports in the literature. See,e.g., Cho et al., 2003, Nature, 421: 756-60; Eigenbrot et al., 2010,PNAS, 107: 15039-44; and Franklin et al., 2004, Caner Cell, 5: 17-28.

The 7C2 Fab binds to the loop 163-175 and the loop 185-189 within theHER2 domain I (i.e., amino acids 163-175 and 185-189 of mature HER2,e.g., SEQ ID NO: 39; domain I is shown in SEQ ID NO: 35). The bindingburies ˜1160 Å² of solvent accessible surface area on each side of theinterface. There is an intricate network of hydrophobic, hydrogenbonding and ionic interactions. Certain residues that are involved inbinding are labeled in FIG. 11C. The side chain of His171 makes contactswith the heavy chain residues His52 and Asp55. The HER2 residues Ser186,Ser187 and Glu188 form hydrogen bonding with the D102 from heavy chainand the two Tyr residues (Tyr36 and Tyr54) from the light chain.

The 7C2 binding epitope partially overlaps with that from a previouslyreported anti-HER2 antibody, chA21 (FIG. 11D). See Zhou et al., 2011,JBC, 286: 31676-83. Both epitopes include a loop in domain I (residues163-187). Interestingly, the residue His171 plays a role in theinteraction with both antibodies. However, the chA21 binding epitopespans ˜1820 Å² of solvent accessible surface area, which is ˜660 Å²bigger than the 7C2 epitope and includes two additional N-terminalloops, residues 100-105 and residues 135-144.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

Table of Sequences SEQ NAME SEQUENCE ID NO Human Her2MELAALCRWG LLLALLPPGA ASTQVCTGTD MKLRLPASPE THLDMLRHLY 1 precursorQGCQVVQGNL ELTYLPTNAS LSFLQDIQEV QGYVLIAHNQ VRQVPLQRLR (UniProtKB/IVRGTQLFED NYALAVLDNG DPLNNTTPVT GASPGGLREL QLRSLTEILK Swiss-Prot:GGVLIQRNPQ LCYQDTILWK DIFHKNNQLA LTLIDTNRSR ACHPCSPMCK P04626.1); aa 1-GSRCWGESSE DCQSLTRTVC AGGCARCKGP LPTDCCHEQC AAGCTGPKHS 22 signalDCLACLHFNH SGICELHCPA LVTYNTDTFE SMPNPEGRYT FGASCVTACP sequence; aa 23-YNYLSTDVGS CTLVCPLHNQ EVTAEDGTQR CEKCSKPCAR VCYGLGMEHL 1255 matureREVRAVTSAN IQEFAGCKKI FGSLAFLPES FDGDPASNTA PLQPEQLQVF Her2ETLEEITGYL YISAWPDSLP DLSVFQNLQV IRGRILHNGA YSLTLQGLGISWLGLRSLRE LGSGLALIHH NTHLCFVHTV PWDQLFRNPH QALLHTANRPEDECVGEGLA CHQLCARGHC WGPGPTQCVN CSQFLRGQEC VEECRVLQGLPREYVNARHC LPCHPECQPQ NGSVTCFGPE ADQCVACAHY KDPPFCVARCPSGVKPDLSY MPIWKFPDEE GACQPCPINC THSCVDLDDK GCPAEQRASPLTSIISAVVG ILLVVVLGVV FGILIKRRQQ KIRKYTMRRL LQETELVEPLTPSGAMPNQA QMRILKETEL RKVKVLGSGA FGTVYKGIWI PDGENVKIPVAIKVLRENTS PKANKEILDE AYVMAGVGSP YVSRLLGICL TSTVQLVTQLMPYGCLLDHV RENRGRLGSQ DLLNWCMQIA KGMSYLEDVR LVHRDLAARNVLVKSPNHVK ITDFGLARLL DIDETEYHAD GGKVPIKWMA LESILRRRFTHQSDVWSYGV TVWELMTFGA KPYDGIPARE IPDLLEKGER LPQPPICTIDVYMIMVKCWM IDSECRPRFR ELVSEFSRMA RDPQRFVVIQ NEDLGPASPLDSTFYRSLLE DDDMGDLVDA EEYLVPQQGF FCPDPAPGAG GMVHHRHRSSSTRSGGGDLT LGLEPSEEEA PRSPLAPSEG AGSDVFDGDL GMGAAKGLQSLPTHDPSPLQ RYSEDPTVPL PSETDGYVAP LTCSPQPEYV NQPDVRPQPPSPREGPLPAA RPAGATLERP KTLSPGKNGV VKDVFAFGGA VENPEYLTPQGGAAPQPHPP PAFSPAFDNL YYWDQDPPER GAPPSTFKGT PTAENPEYLG LDVPVmature human TQVCTGTD MKLRLPASPE THLDMLRHLY QGCQVVQGNL ELTYLPTNAS 39HER2 LSFLQDIQEV QGYVLIAHNQ VRQVPLQRLR IVRGTQLFED NYALAVLDNGDPLNNTTPVT GASPGGLREL QLRSLTEILK GGVLIQRNPQ LCYQDTILWKDIFHKNNQLA LTLIDTNRSR ACHPCSPMCK GSRCWGESSE DCQSLTRTVCAGGCARCKGP LPTDCCHEQC AAGCTGPKHS DCLACLHFNH SGICELHCPALVTYNTDTFE SMPNPEGRYT FGASCVTACP YNYLSTDVGS CTLVCPLHNQEVTAEDGTQR CEKCSKPCAR VCYGLGMEHL REVRAVTSAN IQEFAGCKKIFGSLAFLPES FDGDPASNTA PLQPEQLQVF ETLEEITGYL YISAWPDSLPDLSVFQNLQV IRGRILHNGA YSLTLQGLGI SWLGLRSLRE LGSGLALIHHNTHLCFVHTV PWDQLFRNPH QALLHTANRP EDECVGEGLA CHQLCARGHCWGPGPTQCVN CSQFLRGQEC VEECRVLQGL PREYVNARHC LPCHPECQPQNGSVTCFGPE ADQCVACAHY KDPPFCVARC PSGVKPDLSY MPIWKFPDEEGACQPCPINC THSCVDLDDK GCPAEQRASP LTSIISAVVG ILLVVVLGVVFGILIKRRQQ KIRKYTMRRL LQETELVEPL TPSGAMPNQA QMRILKETELRKVKVLGSGA FGTVYKGIWI PDGENVKIPV AIKVLRENTS PKANKEILDEAYVMAGVGSP YVSRLLGICL TSTVQLVTQL MPYGCLLDHV RENRGRLGSQDLLNWCMQIA KGMSYLEDVR LVHRDLAARN VLVKSPNHVK ITDFGLARLLDIDETEYHAD GGKVPIKWMA LESILRRRFT HQSDVWSYGV TVWELMTFGAKPYDGIPARE IPDLLEKGER LPQPPICTID VYMIMVKCWM IDSECRPRFRELVSEFSRMA RDPQRFVVIQ NEDLGPASPL DSTFYRSLLE DDDMGDLVDAEEYLVPQQGF FCPDPAPGAG GMVHHRHRSS STRSGGGDLT LGLEPSEEEAPRSPLAPSEG AGSDVFDGDL GMGAAKGLQS LPTHDPSPLQ RYSEDPTVPLPSETDGYVAP LTCSPQPEYV NQPDVRPQPP SPREGPLPAA RPAGATLERPKTLSPGKNGV VKDVFAFGGA VENPEYLTPQ GGAAPQPHPP PAFSPAFDNLYYWDQDPPER GAPPSTFKGT PTAENPEYLG LDVPV Murine 7C2.B9DIVLTQSPAS LVVSLGQRAT ISCRASQSVS GSRFTYMHWY QQKPGQPPKL 2 (mu7C2) lightLIKYASILES GVPARFSGGG SGTDFTLNIH PVEEDDTATY YCQHSWEIPP chain variableWTFGGGTKLE IK region Mu7C2 heavyQVQLQQPGAE LVRPGASVKL SCKASGYSFT GYWMNWLKQR PGQGLEWIGM 3 chain variableIHPSDSEIRA NQKFRDKATL TVDKSSTTAY MQLSSPTSED SAVYYCARGT regionYDGGFEYWGQ GTTLTVSS Mu7C2 HVR-L1 RASQSVSGSRFTYMH 4 Mu7C2 HVR-L2 YASILES5 Mu7C2 HVR-L3 QHSWEIPPWT 6 Mu7C2 HVR-H1 GYWMN 7 Mu7C2 HVR-H2MIHPSDSEIRANQKFRD 8 Mu7C2 HVR-H3 GTYDGGFEY 9 HumanizedDIVMTQSPDS LAVSLGERAT INCRASQSVS GSRFTYMHWY QQKPGQPPKL 10 7C2.v2.2.LALIKYASILES GVPDRFSGSG SGTDFTLTIS SLQAEDVAVY YCQHSWEIPP (“hu7C2”) lightWTFGQGTKVE IK chain variable region Hu7C2 heavyEVQLVQSGAE VKKPGASVKV SCKASGYSFT GYWMNWVRQA PGQGLEWIGM 11 chain variableIHPLDAEIRA NQKFRDRVTI TVDTSTSTAY LELSSLRSED TAVYYCARGT regionYDGGFEYWGQ GTLVTVSS Hu7C2 HVR-L1 RASQSVSGSRFTYMH 12 Hu7C2 HVR-L2 YASILES13 Hu7C2 HVR-L3 QHSWEIPPWT 14 Hu7C2 HVR-H1 GYWMN 15 Hu7C2 HVR-H2MIHPLDAEIRANQKFRD 16 (Hu7C2.v2.1.S53L, S55A HVR-H2) Hu7C2 HVR-H3GTYDGGFEY 17 HumanizedDIVMTQSPDS LAVSLGERAT INCRASQSVS GSRFTYMHWY QQKPGQPPKL 18 7C2.v2.2.LALIKYASILES GVPDRFSGSG SGTDFTLTIS SLQAEDVAVY YCQHSWEIPP (hu7C2) kappaWTFGQGTKVE IKRTVAAPSV FIFPPSDEQL KSGTASVVCL LNNFYPREAK light chainVQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACEVTHQGLSSPV TKSFNRGEC  Hu7C2IgG1EVQLVQSGAE VKKPGASVKV SCKASGYSFT GYWMNWVRQA PGQGLEWIGM 19 heavy chainIHPLDAEIRA NQKFRDRVTI TVDTSTSTAY LELSSLRSED TAVYYCARGTYDGGFEYWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDYFPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYICNVNHKPSNT KVDKKVEPKS CDKTHTCPPC PAPELLGGPS VFLFPPKPKDTLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNSTYRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVYTLPPSREEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLDSDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK Hu7C2.v2.1.S53MMIHPMDSEIRANQKFRD 20 HVR-H2 Hu7C2.v2.1.S53L MIHPLDSEIRANQKFRD 21 HVR-H2Hu7C2.v2.1.E101K GTYDGGFKY 22 HVR-H3 HumanizedDIVMTQSPDS LAVSLGERAT INCRASQSVS GSRFTYMHWY QQKPGQPPKL 23 7C2.v2.2.LALIKYASILES GVPDRFSGSG SGTDFTLTIS SLQAEDVAVY YCQHSWEIPP (hu7C2) K149CWTFGQGTKVE IKRTVAAPSV FIFPPSDEQL KSGTASVVCL LNNFYPREAK kappa light chainVQWCVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACEVTHQGLSSPV TKSFNRGEC Hu7C2 A118CEVQLVQSGAE VKKPGASVKV SCKASGYSFT GYWMNWVRQA PGQGLEWIGM 24IgG1 heavy chain IHPLDAEIRA NQKFRDRVTI TVDTSTSTAY LELSSLRSED TAVYYCARGTYDGGFEYWGQ GTLVTVSSCS TKGPSVFPLA PSSKSTSGGT AALGCLVKDYFPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYICNVNHKPSNT KVDKKVEPKS CDKTHTCPPC PAPELLGGPS VFLFPPKPKDTLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNSTYRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVYTLPPSREEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLDSDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK V205C cysteineTVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN 25engineered light SQESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPCTKSchain constant FNRGEC region (Igκ) A118C cysteineCSTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV 26engineered heavy HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEPchain constant KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVSregion (IgG1) HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGKEYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTKNQVSLTCLVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRWQQGNVFSCSV MHEALHNHYT QKSLSLSPGK K149C cysteineTVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW CVDNALQSGN 27engineered light SQESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKSchain constant FNRGEC region (Igκ) S400C cysteineASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV 28engineered heavy HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEPchain constant KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVSregion (IgG1) HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGKEYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTKNQVSLTCLVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDCDGSFFLY SKLTVDKSRWQQGNVFSCSV MHEALHNHYT QKSLSLSPGK

1. An immunoconjugate comprising an antibody and a cytotoxic agent,wherein the immunoconjugate has the formula Ab-(L-D)p, wherein: (a) Abis the antibody; (b) L is a linker; (c) D is a cytotoxic agent; and (d)p ranges from 1-8; and wherein D is a pyrrolobenzodiazepine and L-Dcomprises the structure:

wherein: Y has the formula:

G is a linker connected to the antibody; n is an integer selected in therange of 0 to 48; and wherein the antibody is a humanized monoclonalantibody that binds HER2 comprising (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO: 15; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO: 16; (c) HVR-H3 comprising the amino acid sequence of SEQID NO: 17; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:12; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and(f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:14.
 2. Theimmunoconjugate of claim 1, wherein the antibody comprises a heavy chainvariable region comprising the sequence of SEQ ID NO: 11 and a lightchain variable region comprising the sequence of SEQ ID NO:
 10. 3.(canceled)
 4. (canceled)
 5. The immunoconjugate of claim 1, which is anantibody fragment that binds HER2.
 6. The immunoconjugate of claim 1,wherein HER2 is human HER2 comprising amino acids 23 to 1255 of SEQ IDNO:
 1. 7. The immunoconjugate of an claim 1, wherein the antibody bindsto extracellular domain I of HER2.
 8. The immunoconjugate of claim 7,wherein extracellular domain I of HER2 has the sequence of SEQ ID NO:35.
 9. The immunoconjugate of claim 4, which is an IgG1, IgG2a or IgG2bantibody.
 10. The immunoconjugate of claim 1, wherein the antibodycomprises one or more engineered free cysteine amino acids residues. 11.The immunoconjugate of claim 10, wherein the one or more engineered freecysteine amino acids residues are located in the heavy chain.
 12. Theimmunoconjugate of claim 10, wherein the one or more engineered freecysteine amino acids residues are located in the light chain.
 13. Theimmunoconjugate of claim 11, wherein the antibody comprises at least onemutation in the heavy chain constant region selected from A118C andS400C.
 14. The immunoconjugate of claim 12, wherein the antibodycomprises at least one mutation in the light chain constant regionselected from K149C and V205C.
 15. The immunoconjugate of claim 1,wherein the antibody comprises: a) a heavy chain comprising the sequenceof SEQ ID NO: 19 and a light chain comprising the sequence of SEQ ID NO:18; or b) a heavy chain comprising the sequence of SEQ ID NO: 19 and alight chain comprising the sequence of SEQ ID NO: 23; or c) a heavychain comprising the sequence of SEQ ID NO: 24 and a light chaincomprising the sequence of SEQ ID NO:
 18. 16. The immunoconjugate ofclaim 1, wherein the antibody comprises the heavy chain constant regionof SEQ ID NO:
 28. 17. The immunoconjugate of claim 1, wherein theantibody comprises the light chain constant region of SEQ ID NO: 25.18.-32. (canceled)
 33. The immunoconjugate of claim 1, comprising thestructure:


34. The immunoconjugate of claim 1, wherein p ranges from 1.3-2 or from2-5.
 35. A pharmaceutical formulation comprising the immunoconjugate ofclaim 1 and a pharmaceutically acceptable carrier.
 36. Thepharmaceutical formulation of claim 35, further comprising an additionaltherapeutic agent.
 37. The pharmaceutical formulation of claim 36,wherein the additional therapeutic agent is an antibody orimmunoconjugate that binds HER2.
 38. The pharmaceutical formulation ofclaim 37, wherein the additional therapeutic agent is (i) an antibody orimmunoconjugate that binds to domain II of HER2, and/or (ii) an antibodyor immunoconjugate that binds to domain IV or HER2.
 39. Thepharmaceutical formulation of claim 38, wherein the additionaltherapeutic agent is (i) an antibody or immunoconjugate that binds toepitope 2C4, and/or (ii) an antibody or immunoconjugate that binds toepitope 4D5.
 40. The pharmaceutical formulation of claim 36, wherein theadditional therapeutic agent is trastuzumab, trastuzumab-MCC-DM1(T-DM1), and/or pertuzumab.
 41. The pharmaceutical formulation of claim36, further comprising (1) trastuzumab or T-DM1, and (2) pertuzumab. 42.A method of treating an individual having a HER2-positive cancer, themethod comprising administering to the individual an effective amount ofthe immunoconjugate of claim
 1. 43. The method of claim 42, wherein theHER2-positive cancer is breast cancer or gastric cancer.
 44. The methodof claim 43, wherein the HER2-positive breast cancer is early-stagebreast cancer.
 45. The method of claim 43, wherein the HER2-positivebreast cancer is metastatic breast cancer.
 46. The method of claim 42,further comprising administering an additional therapeutic agent to theindividual.
 47. A method of treating an individual having aHER2-positive cancer, the method comprising administering to theindividual an effective amount of the immunoconjugate of claim 1 and atleast one additional therapeutic agent to the individual.
 48. The methodof claim 47, wherein the additional therapeutic agent is an antibody orimmunoconjugate that binds HER2.
 49. The method of claim 48, wherein theadditional therapeutic agent is (i) an antibody or immunoconjugate thatbinds to domain II of HER2, and/or (ii) an antibody or immunoconjugatethat binds to domain IV or HER2.
 50. The method of claim 49, wherein theadditional therapeutic agent is (i) an antibody or immunoconjugate thatbinds to epitope 2C4, and/or (ii) an antibody or immunoconjugate thatbinds to epitope 4D5.
 51. The method of claim 47, wherein the additionaltherapeutic agent is selected from trastuzumab, trastuzumab-MCC-DM1(T-DM1), and pertuzumab.
 52. The method of claim 47, wherein theadditional therapeutic agents are (1) trastuzumab or T-DM1, and (2)pertuzumab.
 53. The method of claim 47, wherein the HER2-positive canceris breast cancer or gastric cancer.
 54. The method of claim 53, whereinthe HER2-positive breast cancer is metastatic breast cancer.
 55. Themethod of claim 53, wherein the HER2-positive breast cancer isearly-stage breast cancer.
 56. The method of claim 42, wherein theHER2-positive cancer is recurrent cancer.
 57. The method of claim 56,wherein the recurrent cancer is locally recurrent cancer.
 58. The methodof claim 42, wherein the HER2-positive cancer is advanced cancer. 59.The method of claim 42, wherein the HER2-positive cancer isnon-resectable.
 60. A method of treating an individual having aHER2-positive cancer, comprising: a) subjecting the individual toneoadjuvant treatment with the immunoconjugate claim 1, b) removing thecancer by definitive surgery, and c) subjecting the individual toadjuvant treatment with the immunoconjugate of claim
 1. 61. The methodof claim 60, wherein the HER2-positive cancer is breast cancer orgastric cancer.
 62. The method of claim 61, wherein the HER2-positivecancer is breast cancer.
 63. A method of inhibiting proliferation of aHER2-positive cell, the method comprising exposing the cell to theimmunoconjugate of claim 1 under conditions permissive for binding ofthe immunoconjugate to HER2 on the surface of the cell, therebyinhibiting proliferation of the cell.
 64. The method of claim 63,wherein the cell is a breast cancer cell or a gastric cancer cell.